Lipolytic enzyme variants

ABSTRACT

The present disclosure describes a variant polypeptide having lipolytic activity, wherein the variant has an amino acid sequence which, when aligned with the amino acid sequence as set out in SEQ ID NO: 2, comprises at least one substitution of an amino acid residue at a position corresponding to any of the positions 53, 112, 141, 178, 179, 182, 202, 203, 235, 282, 284, 
     said positions being defined with reference to SEQ ID NO: 2, and wherein said variant has at least 70% identity with the mature polypeptide having lipolytic activity as set out in SEQ ID NO: 2. 
     Such a variant polypeptide may be used in the preparation of a baked product.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage entry of InternationalApplication No. PCT/EP2017/083527, filed 19 Dec. 2017, which claimspriority to European Patent Application No. 16205899.4, filed 21 Dec.2016, European Patent Application No. 16205906.7, filed 21 Dec. 2016,European Patent Application No. 17157058.3, filed 21 Feb. 2017, EuropeanPatent Application No. 17157061.7, filed 21 Feb. 2017, and EuropeanPatent Application No. 17157069.0, filed 21 Feb. 2017.

Reference to Sequence Listing Submitted as a Compliant ASCII Text File(.txt)

Pursuant to the EFS-Web legal framework and 37 C.F.R. § 1.821-825 (seeM.P.E.P. § 2442.03(a)), a Sequence Listing in the form of anASCII-compliant text file (entitled“2919208-510000_Sequence_Listing_ST25.txt” created on 3 Jun. 2019, and24,632 bytes in size) is submitted concurrently with the instantapplication, and the entire contents of the Sequence Listing areincorporated herein by reference.

BACKGROUND Field

The present disclosure relates to a variant polypeptide having lipolyticactivity, also referred to as a lipolytic enzyme variant. The disclosurealso relates to a nucleic acid sequence encoding such a variant, anucleic acid construct comprising the nucleic acid sequence and torecombinant host cell comprising a recombinant expression vectorcomprising said nucleic acid construct encoding the variant. Further,the disclosure relates to a method for producing a lipolytic polypeptidevariant. The disclosure further relates to a composition comprising thevariant polypeptide, use of such variant polypeptide in the productionof a food product and to use of the variant polypeptide to replace atleast part of a chemical emulsifier in the production of a dough and/ora baked product. The disclosure further relates to a dough, a processfor the production of a dough and a process for the production of abaked product.

Description of Related Art

In the baking industry, e.g. in the industrial dough and bread making,processing aids are used to improve properties of a dough and or a bakedproduct. In order to improve the handling properties of the dough and/orthe final properties of the baked products there is a continuous effortto develop processing aids with improved properties. Processing aids aredefined herein as compounds that improve the handling properties of thedough and/or the final properties of the baked products. Doughproperties that may be improved comprise stability, gas retainingcapability, elasticity, extensibility, moldability etcetera. Propertiesof the baked products that may be improved comprise loaf volume, crustcrispiness, oven spring, crumb texture, crumb structure, crumb softness,flavour, relative staleness and shelf life. These processing aids may bedivided into two groups: chemical additives and enzymes (also referredto as baking enzymes).

Chemical additives with improving properties include oxidising agentssuch as ascorbic acid, bromate and azodicarbonate, reducing agents suchas L-cysteine and glutathione, emulsifiers acting as dough conditionerssuch as diacetyl tartaric acid esters of mono/diglycerides (DATEM),sodium stearoyl lactylate (SSL) or calcium stearoyl lactylate (CSL),emulsifiers acting as crumb softeners such as glycerol monostearate(GMS) etceteras, fatty materials such as triglycerides (fat) or lecithinand others. Emulsifiers such as DATEM, SSL and/or CSL may be used forgenerating process tolerance. Emulsifiers may also be used to increasevolume of a baked product. The resistance of consumers to chemicaladditives is growing and there is therefore constant need to replacechemical additives such as chemical emulsifiers.

There are currently replacers of chemical emulsifiers in the market suchas lipolytic enzymes that upon action of a substrate can generateemulsifying molecules in situ. Lipolytic enzymes are enzymes thatcatalyse the hydrolysis of ester bonds in lipid substrates, leading tothe release of fatty acids. In industry, phospholipases are used tofully or partly replace e.g. DATEM. WO1998026057 describes aphospholipase that can be used in a process for making bread.WO2009/106575 describes a lipolytic enzyme and its use in a process formaking bread. Despite the fact that there are commercial lipolyticenzymes in the market there is still an industrial need for lipolyticenzymes with improved performance in industry, especially in the bakingindustry.

DESCRIPTION OF THE FIGURES

FIG. 1. Sets out the Aspergillus expression vector pGBFIN-50.

FIG. 2. Sets out the Aspergillus lipolytic enzyme expression vectorpGBFINPL-00

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 sets out the polynucleotide sequence encoding the referencepolypeptide having lipolytic activity (set out in nucleotides 100 to914) including an N-terminal signal sequence of 33 amino acids (set outin nucleotides 1 to 99), and C-terminal pro-sequence (set out innucleotides 915 to 1038).

SEQ ID NO: 2 sets out the amino acid sequence of the referencepolypeptide (also referred to as parent polypeptide) having lipolyticactivity (set out in amino acids 34 to 304) including an N-terminalsignal sequence of 33 amino acids (set out in amino acids 1 to 33), andC-terminal pro-sequence (set out in amino acids 305 to 346).

SEQ ID NO: 3 sets out a modified translational initiation sequence ofthe glucoamylase glaA promoter.

SEQ ID NO: 4 sets out the amino acid sequence of an Alicyclobacilluspohliae alpha-amylase polypeptide.

SEQ ID NO: 5 sets out the amino acid sequence of a Bacillusstearothermophilus amylase polypeptide.

SEQ ID NO: 6 sets out the amino acid sequence of an amylase fromPseudomonas saccharophila

SEQ ID NO: 7 sets out the amino acid sequence of an amylase.

SUMMARY

According to the disclosure there is provided a variant polypeptidehaving lipolytic activity, wherein the variant has an amino acidsequence which, when aligned with the amino acid sequence as set out inSEQ ID NO: 2, comprises at least one substitution of an amino acidresidue at a position corresponding to any of the positions 53, 112,141, 178, 179, 182, 202, 203, 235, 282, 284,

said positions being defined with reference to SEQ ID NO: 2,

and wherein said variant has at least 70% identity with the maturepolypeptide having lipolytic activity as set out in SEQ ID NO: 2.

The disclosure also provides:

-   -   a nucleic acid sequence encoding the variant polypeptide of the        disclosure;    -   A nucleic acid construct comprising the nucleic acid sequence        operably linked to one or more control sequences capable of        directing the expression of a lipolytic enzyme in a suitable        expression host.    -   A recombinant host cell comprising a recombinant expression        vector comprising the nucleic acid construct.

The disclosure also relates a method for producing a lipolyticpolypeptide variant comprising cultivating the host cell underconditions conducive to production of the lipolytic enzyme variant andrecovering the lipolytic enzyme variant.

Further the disclosure relates to:

-   -   A composition comprising the variant polypeptide of the        disclosure;    -   Use of the variant polypeptide according to the disclosure, or        of the composition according to the disclosure in the production        of a food product, preferably in the production of a dough        and/or a baked product    -   A dough comprising the variant polypeptide according to the        disclosure, a variant polypeptide obtainable by the method        according to the disclosure or the composition according to the        disclosure.    -   A process for the production of a dough comprising the step of        combining an effective amount of the variant polypeptide        according to the disclosure, the variant polypeptide obtainable        by the method according to the disclosure or the composition        according to the disclosure to at least one dough ingredient.    -   A process for the production of a baked product, which method        comprises baking the dough according to the disclosure.

DETAILED DESCRIPTION

Throughout the present specification and the accompanying claims, thewords “comprise”, “include” and “having” and variations such as“comprises”, “comprising”, “includes” and “including” are to beinterpreted inclusively. That is, these words are intended to convey thepossible inclusion of other elements or integers not specificallyrecited, where the context allows.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to one or at least one) of the grammatical object of thearticle. By way of example, “an element” may mean one element or morethan one element.

The present disclosure concerns variant polypeptides having lipolyticactivity. The variant polypeptides according to the disclosure have atleast one altered property as compared with a reference polypeptidehaving lipolytic activity. In an aspect of the variant polypeptide, thereference polypeptide comprises the mature lipolytic enzyme as set outin SEQ ID NO: 2. In a further aspect of the variant polypeptide, thereference polypeptide is the mature lipolytic enzyme as set out in SEQID NO: 2.

The variant has at least 70% identity with the mature polypeptide havinglipolytic activity as set out in SEQ ID NO: 2. In an aspect of thevariant polypeptide, the mature polypeptide comprises an amino acidsequence as set out in amino acids 34 to 304 of SEQ ID NO: 2. In afurther aspect of the variant polypeptide, the mature polypeptide has anamino acid sequence as set out in amino acids 34 to 304 of SEQ ID NO: 2.

The reference polypeptide may also be referred to herein as a parentpolypeptide or comparison polypeptide.

A variant polypeptide according to the disclosure may be an isolated,substantially pure, pure, recombinant or synthetic polypeptide.

In an embodiment, the variant polypeptide according to the disclosure isa non-naturally occurring polypeptide.

Herein, positions which may be substituted to achieve a variant of thedisclosure are defined with reference to SEQ ID NO: 2. The variant hasan amino acid sequence which, when aligned with the amino acid sequenceas set out in SEQ ID NO: 2, comprises at least one substitution of anamino acid residue at a position corresponding to any of the positions53, 112, 141, 178, 179, 182, 202, 203, 235, 282, 284,

said positions being defined with reference to SEQ ID NO: 2.

More concretely, the disclosure relates to a variant polypeptide havinglipolytic activity, wherein the variant has an amino acid sequencewhich, when aligned with the amino acid sequence as set out in SEQ IDNO: 2, comprises at least one substitution of an amino acid residue at aposition corresponding to any of the positions 53, 112, 141, 178, 179,182, 202, 203, 235, 282, 284, said positions being defined withreference to SEQ ID NO: 2,

and wherein said variant has at least 70% identity with the maturepolypeptide having lipolytic activity as set out in SEQ ID NO: 2.

In an embodiment the disclosure relates to a variant polypeptide havinglipolytic activity, wherein the variant has an amino acid sequencewhich, when aligned with the amino acid sequence as set out in SEQ IDNO: 2, comprises at least one substitution of an amino acid residue at aposition corresponding to any of the positions 53, 112, 141, 178, 179,182, 202, 203, 235, 282, 284,

said positions being defined with reference to SEQ ID NO: 2,

and wherein the variant has one or more altered properties as comparedwith a reference polypeptide having lipolytic activity and wherein saidvariant has at least 70% identity with the mature polypeptide havinglipolytic activity as set out in SEQ ID NO: 2.

In an embodiment the mature polypeptide comprises an amino acid sequenceas set out in amino acids 34 to 304 of SEQ ID NO: 2. In an embodimentthe reference polypeptide having lipolytic activity comprises an aminoacid sequence as set out in amino acids 34 to 304 of SEQ ID NO: 2. In afurther embodiment such variant has one or more altered properties ascompared to the reference polypeptide having lipolytic activity as setout in amino acids 34 to 304 of SEQ ID NO: 2 measured under the sameconditions, and said variant has at least 70% identity with the maturepolypeptide having lipolytic activity as set out in amino acids 34 to304 of SEQ ID NO: 2.

In an embodiment the variant polypeptide according to the disclosure hasan amino acid sequence which, when aligned with the amino acid sequenceas set out in SEQ ID NO: 2, comprises at least one substitution of anamino acid residue at a position corresponding to any of the positions53, 112, 141, 178, 179, 182, 202, 203, 235, 282, 284,

said positions being defined with reference to SEQ ID NO: 2,

and wherein said variant has at least 70% identity with the maturepolypeptide having lipolytic activity as set out in SEQ ID NO: 2.

In an embodiment, the variant polypeptide according to the disclosurehas an amino acid sequence which, when aligned with the amino acidsequence as set out in SEQ ID NO: 2, comprises at least one substitutionof an amino acid residue at a position corresponding to any of thepositions 53, 112, 141, 178, 179, 182, 202, 203, 235, 282, 284,

said positions being defined with reference to SEQ ID NO: 2,

and wherein the variant has one or more altered properties as comparedwith a reference polypeptide having lipolytic activity and wherein saidvariant has at least 70% identity with the mature polypeptide havinglipolytic activity as set out in SEQ ID NO: 2.

In an embodiment, the variant polypeptide according to the disclosurehas an amino acid sequence which, when aligned with the amino acidsequence as set out in SEQ ID NO: 2, comprises at least one substitutionof an amino acid residue at a position corresponding to any of thepositions 53, 112, 141, 178, 179, 182, 202, 203, 235, 282, 284,

said positions being defined with reference to SEQ ID NO: 2,

and wherein the variant has an altered ratio of the activity on estersof long chain fatty acids: the activity on esters of short chain fattyacids as compared with a reference polypeptide having lipolytic activityand wherein said variant has at least 70% identity with the maturepolypeptide having lipolytic activity as set out in SEQ ID NO: 2.

In an embodiment, the variant polypeptide according to the disclosurehas an amino acid sequence which, when aligned with the amino acidsequence as set out in SEQ ID NO: 2, comprises at least one substitutionof an amino acid residue at a position corresponding to any of thepositions

53, 112, 141, 178, 179, 182, 202, 203, 235, 282, 284,

said positions being defined with reference to SEQ ID NO: 2,

and the variant has an altered ratio of the activity on esters of longchain fatty acids: the activity on esters of short chain fatty acids ascompared with a reference polypeptide having lipolytic activity as setout in amino acids 34 to 304 of SEQ ID NO: 2 and said variant has atleast 70% identity with the mature polypeptide having lipolytic activityas set out in amino acids 34 to 304 of SEQ ID NO: 2.

In an embodiment, the variant polypeptide according to the disclosurehas an amino acid sequence which, when aligned with the amino acidsequence as set out in SEQ ID NO: 2, comprises at least one substitutionof an amino acid residue at a position corresponding to any of thepositions 53, 112, 141, 178, 179, 182, 202, 203, 235, 282, 284,

said positions being defined with reference to SEQ ID NO: 2,

and wherein the variant has an increased ratio of the activity on estersof long chain fatty acids: the activity on esters of short chain fattyacids as compared with a reference polypeptide having lipolytic activityand wherein said variant has at least 70% identity with the maturepolypeptide having lipolytic activity as set out in SEQ ID NO: 2.

In an embodiment, the variant polypeptide according to the disclosurehas an amino acid sequence which, when aligned with the amino acidsequence as set out in SEQ ID NO: 2, comprises at least one substitutionof an amino acid residue at a position corresponding to any of thepositions

53, 112, 141, 178, 179, 182, 202, 203, 235, 282, 284,

said positions being defined with reference to SEQ ID NO: 2,

and the variant has an increased ratio of the activity on esters of longchain fatty acids: the activity on esters of short chain fatty acids ascompared with a reference polypeptide having lipolytic activity as setout in amino acids 34 to 304 of SEQ ID NO: 2 and said variant has atleast 70% identity with the mature polypeptide having lipolytic activityas set out in amino acids 34 to 304 of SEQ ID NO: 2.

In an aspect the lipolytic enzyme variant according to the disclosure,is a lipolytic enzyme variant having at least 70% identity, in an aspectat least 75% identity, in an aspect at least 80% identity, in an aspectat least 85% identity, in an aspect at least 90% identity, in an aspectat least 95% identity, in an aspect at least 96% identity, in an aspectat least 97% identity, in an aspect at least 98% identity, in an aspectat least 99% identity, with the mature polypeptide having lipolyticactivity as set out in the amino acids sequence of SEQ ID NO: 2.

In an aspect the lipolytic enzyme variant according to the disclosure,is a lipolytic enzyme variant having at least 70% identity, in an aspectat least 75% identity, in an aspect at least 80% identity, in an aspectat least 85% identity, in an aspect at least 90% identity, in an aspectat least 95% identity, in an aspect at least 96% identity, in an aspectat least 97% identity, in an aspect at least 98% identity, in an aspectat least 99% identity, with the mature polypeptide having lipolyticactivity as set out in amino acids 34 to 304 of SEQ ID NO: 2.

The lipolytic enzyme variant of the disclosure may comprise one or moresubstitutions at the positions disclosed herein. The lipolytic enzymevariant of the disclosure may for example comprise two, at least two, atleast three, at least four, at least 5, at least 10, at least 15 or atleast 20 of the disclosed positions.

The lipolytic enzyme variant of the disclosure may comprise one or morefurther substitutions not disclosed herein as long as the variant hasone or more altered properties, as described herein, as compared with areference polypeptide having lipolytic activity. The one or more furthersubstitutions may be selected from any of the positions 53, 112, 113,117, 122, 124, 138, 141, 178, 179, 182, 200, 202, 203, 229, 238, 282,284, 286, 295, said positions being defined with reference to SEQ ID NO:2. Preferred substitutions are listed below (with the positions beingdefined in relation to the sequence set out in SEQ ID NO: 2). A variantof the disclosure may be generated using any combination ofsubstitutions listed below or in Table 1.

A “substitution” in this context indicates that a position in thevariant which corresponds to one of the positions set out above in SEQID NO: 2 comprises an amino acid residue which does not appear at thatposition in the reference polypeptide having lipolytic activity

Amino acids changes are depicted according to the single letterannotation.

The disclosure provides a variant polypeptide according to thedisclosure, wherein the reference polypeptide comprises an amino acidsequence as set out in amino acids 34 to 304 of SEQ ID NO: 2 and whereinthe mature polypeptide comprises an amino acid sequence as set out inamino acids 34 to 304 of SEQ ID NO: 2.

In an embodiment, the mature polypeptide has an amino acid sequence asset out in amino acids 34 to 304 of SEQ ID NO: 2. In an embodiment thereference polypeptide having lipolytic activity has an amino acidsequence as set out in amino acids 34 to 304 of SEQ ID NO: 2.

Mature Polypeptide

The lipolytic enzyme variant as the disclosure is a mature polypeptide.

A “mature polypeptide” is defined herein as a polypeptide in its finalform and is obtained after translation of an mRNA into polypeptide andpost-translational modifications of said polypeptide. Post-translationalmodification includes N-terminal processing, C-terminal truncation,glycosylation, phosphorylation and removal of leader sequences such assignal peptides, propeptides and/or prepropeptides by cleavage. Theprocess of maturation may depend on the particular expression vectorused, the expression host and the production process. Preferably, themature polypeptide having lipolytic activity in the amino acid sequenceas set out in SEQ ID NO: 2 is the polypeptide as set out in amino acids34 to 304 of SEQ ID NO: 2.

A “mature polypeptide coding sequence” means a polynucleotide thatencodes a mature polypeptide (with reference to its amino acidsequence).

A “nucleotide sequence encoding a mature polypeptide” is defined hereinas the polynucleotide sequence which codes for a mature polypeptide.

As is known to the person skilled in the art it is possible that theN-terminus of a mature polypeptide might be heterogeneous due toprocessing errors during secretion and/or maturation. As is known to theperson skilled in the art it is possible that the C-terminus of a maturepolypeptide, might be heterogeneous due to processing errors duringsecretion and/or maturation. In particular such processing errors mightoccur upon overexpression of the polypeptide. In addition, exo-proteaseactivity might give rise to heterogeneity. The extent to whichheterogeneity occurs depends also on the host and fermentation protocolsthat are used. Such C-terminal processing artefacts might lead to ashorter or a longer mature polypeptide. Processing errors duringsecretion and/or maturation may also lead to a heterogeneous N-terminus.

In an embodiment, the lipolytic enzyme variant contains one or moreadditional residues and starts at position −1, or −2, or −3 etc. saidposition being defined with reference to position 34 of SEQ ID NO: 2. Insuch event the N terminus starts at position 33, or 32, or 31 etc.

Alternatively, it might lack certain residues and as a consequencestarts at position 2, or 3, or 4 etc. said position being defined withreference to position 34 of SEQ ID NO: 2. In such event the N terminusstarts at position 35, or 36, or 37 etc.

Further, also additional residues may be present at the C-terminus, e.g.the C-terminus ends at position 305, 306 etc. Alternatively, theC-terminus might lack certain residues and as a consequence end atposition 303, or 302 etc.

In an aspect, the mature enzyme comprises the polypeptide as set out inamino acids 34 to 303 of SEQ ID NO: 2.

In an aspect, the mature enzyme comprises the polypeptide as set out inamino acids 31 to 304 of SEQ ID NO: 2.

In an aspect, the mature enzyme comprises the polypeptide as set out inamino acids 31 to 303 of SEQ ID NO: 2.

In an aspect, the mature enzyme comprises the polypeptide as set out inamino acids 34 to 307 of SEQ ID NO: 2.

In an aspect, the mature enzyme comprises the polypeptide as set out inamino acids 34 to 302 of SEQ ID NO: 2.

In an aspect, the mature enzyme comprises the polypeptide as set out inamino acids 31 to 307 of SEQ ID NO: 2.

In an embodiment, the variant polypeptide according to the disclosure,the variant has an amino acid sequence which, when aligned with theamino acid sequence as set out in SEQ ID NO: 2, comprises one or more ofthe amino acid substitutions selected from

-   -   Y53S    -   S112T    -   F141M    -   A178G    -   V179L    -   V179M    -   L182F    -   P202A    -   R203M    -   P235L    -   L282F    -   I284Q        said positions being defined with reference to SEQ ID NO: 2, and        wherein said variant has at least 70% identity with the mature        polypeptide having lipolytic activity as set out in SEQ ID        NO: 2. In a further embodiment such variant has one or more        altered properties as compared with a reference polypeptide        having lipolytic activity. In a further embodiment, such variant        has one or more altered properties as compared to the reference        polypeptide having lipolytic activity as set out in amino acids        34 to 304 of SEQ ID NO: 2 measured under the same conditions,        and said variant has at least 70% identity with the mature        polypeptide having lipolytic activity as set out in amino acids        34 to 304 of SEQ ID NO: 2.

In an embodiment, the variant polypeptide according to the disclosurehas an amino acid sequence which, when aligned with the amino acidsequence as set out in SEQ ID NO: 2, comprises one or more of the aminoacid substitutions selected from

-   -   Y53S    -   S112T    -   F141M    -   A178G    -   V179L    -   V179M    -   L182F    -   P202A    -   R203M    -   P235L    -   L282F    -   I284Q        said positions being defined with reference to SEQ ID NO: 2, and        wherein said variant has at least 70% identity with the mature        polypeptide having lipolytic activity as set out in SEQ ID        NO: 2. In a further embodiment the variant has one or more        altered properties (preferably has an increased ratio of the        activity on esters of long chain fatty acids: the activity on        esters of short chain fatty acids) as compared with a reference        polypeptide having lipolytic activity. In a further embodiment        such variant has an increased ratio of the activity on esters of        long chain fatty acids: the activity on esters of short chain        fatty acids as compared to the reference polypeptide having        lipolytic activity as set out in amino acids 34 to 304 of SEQ ID        NO: 2 measured under the same conditions, and said variant has        at least 70% identity with the mature polypeptide having        lipolytic activity as set out in amino acids 34 to 304 of SEQ ID        NO: 2.

Variant Polypeptide Having Lipolytic Activity

A lipolytic enzyme demonstrates lipolytic activity, i.e. it is able tocatalyse the hydrolysis of ester bonds in lipids, such as triglyceridesand/or galactolipids and/or phospholipids. In particular lipolyticenzyme variants according to the disclosure demonstrate lipolyticactivity on lipids present in flour. Flour includes cereal flour, cornflour, rice flour. Flour herein includes whole-meal flour.

Wheat flour contains approximately 2-3% lipids. The flour lipids can bedivided into starch lipids (0.8-1%) and non-starch lipids (1.4-2.0%).Whereas the starch lipids consist mainly of polar lysophospholipids, thenon-starch lipids consist of about 40% neutral triglycerides and 40%polar phospho- and glycolipids. For optimisation of the flour lipidsfraction the lipolytic polypeptide variant according to the disclosureis in an aspect capable of hydrolysis of the polar lipids, being thephospholipids and glycolipids (more specifically the galactolipids) insitu in the dough by adding the lipolytic enzyme variant according tothe disclosure.

The lipolytic activity of the lipolytic enzyme variants according to thedisclosure may be determined by any suitable method, e.g. by assaysknown in the art or described later herein. A variant polypeptide havinglipolytic activity and lipolytic polypeptide variant, a lipolytic enzymevariant and a variant polypeptide are used interchangeably herein. Apolypeptide having lipolytic activity, a lipolytic polypeptide, and alipolytic enzyme are used interchangeably herein. The variants describedherein are collectively comprised in the terms “a lipolytic polypeptidevariant according to the disclosure” or “a lipolytic enzyme variantaccording to the disclosure” or “a variant polypeptide according to thedisclosure”.

A triacylglycerol lipase (EC 3.1.1.3) demonstrates catalytic hydrolyticactivity on one or more ester bonds in triglycerides (also known astriacylglycerol or triacylglycerides). The wordings “activity ontriacylglycerols (TAG)” and “TAG-lipase activity” are usedinterchangeably herein. The wordings “triglycerides” and“triacylglycerides” and “triacylglycerols” are used interchangeablyherein

In general triglycerides and triglyceride are used interchangeablyherein: they both refer to the compound class.

A galactolipase demonstrates galactolipase activity (EC 3.1.1.26).Galactolipase activity is catalytic hydrolytic activity on one or morebonds in the galactolipids. The wordings “galactolipase activity” and“activity on galactolipids” are used interchangeably herein.

Galactolipids consist of a glycerol backbone with esterified fatty acid,while the third hydroxyl group is bound to sugar residues such as incase of galactolipids a galactose, for example monogalactosyldiglycerideor digalactosyldiglyceride. In general, galactolipids and galactolipidare used interchangeably herein: they both refer to the compound class.In wheat, galactolipids are mainly present as diacylgalactolipids. In anaspect of the disclosure the galactolipids are DGDG(digalactosyldiglycerides) or MGDG (monogalactosyldiglycerides).

A phospholipase demonstrates phospholipase activity. Phospholipaseactivity is a catalytic hydrolytic activity on one or more bonds in thephospholipids. The wordings “phospholipase activity” and “activity onphospholipids” are used interchangeably herein. Phospholipids consist ofa glycerol backbone with esterified fatty acid, while the third hydroxylgroup of the glycerol is esterified with phosphoric acid. The phosphoricacid may, in turn, be esterified to for example an amino alcohol likeethanolamine (phosphatidylethanolamine), choline (phosphatidylcholine).Several types of phospholipase activity can be distinguished whichhydrolyse the ester bond(s) that link the fatty acyl moieties to theglycerol backbone:

-   -   Phospholipase A1 and A2 activity concern the deacylation of one        fatty acyl group in the outer sn-1 and middle sn-2 positions        respectively, from a diacylglycerophospholipid to produce a        lysophospholipid. This is a desirable activity for emulsifier        replacement. Phospholipase A1 has EC number EC 3.1.1.32 and        phospholipase A2 EC 3.1.1.4.    -   Lysophospholipase activity (also called phospholipase B        activity) concerns the hydrolysis of the remaining fatty acyl        group in a lysophospholipid. For emulsifier replacement        lysophospholipase activity is usually less desirable.        Lysophospholipase has EC number EC 3.1.1.5.

In general phospholipids and phospholipid are used interchangeablyherein: they both refer to the compound class. In an aspect of thedisclosure phospholipids are phosphatidylcholines (PC).

Lipolytic enzymes are usually classified according to the bonds thatthey preferentially cleave, but can show some activity beyond their mostpreferred substrates. For example, a PLA1 phospholipase may showgreatest activity towards the cleavage of acyl chains from the sn1position of the glycerol backbone of a phospholipid, but some activityon TAG, galactolipids and sn2 hydrolysis may also occur.

The wordings “ratio of the activity on galactolipids: the activity ontriacylglycerols” and “galactolipase: TAG-lipase activity ratio” and“galactolipase to TAG-lipase activity ratio” and “ratio of thegalactolipase activity: the TAG-lipase activity” are usedinterchangeably herein.

The wordings “ratio of the activity on phospholipids: the activity ontriacylglycerols” and “phospholipase: TAG-lipase activity ratio” and“phospholipase to TAG-lipase activity ratio” and “ratio of thephospholipase activity: the TAG-lipase activity” are usedinterchangeably herein.

Polar lipids herein include phospholipids and galactolipids.

Non-polar lipids herein include triglycerides and/or diglycerides. Thenon-polar lipid can be triolein.

Herein esters of long chain fatty acids are esters of C12-C18 fattyacids, for instance esters of C16-C18 fatty acids, for instance estersof C18 fatty acids, for instance esters of C18:2 fatty acids.

Herein esters of short chain fatty acids are esters of C4-C8 fattyacids, for instance esters of C4-C6 fatty acids, in an aspect esters ofC4 fatty acids.

The ester of the short chain fatty can be pNP-butyrate.

The ester of the long chain fatty can be pNP-lineolate, pNP-oleate,pNP-stearate or pNP-palmitate.

The ester of the non-saturated chain fatty is: pNP-lineolate orpNP-oleate.

The ester of the saturated chain fatty is: pNP-stearate orpNP-palmitate.

Altered/Improved Property

A variant polypeptide according to the disclosure will typically have analtered property as compared to a reference polypeptide. In particularthe variant polypeptide will have an improved property as compared to areference polypeptide which is relevant to the use of the variantpolypeptide in the food industry, preferably in the preparation of adough and/or a baked product.

The altered, typically improved, property may be demonstrated by makinga dough and/or baked product comprising the lipolytic enzyme variant ofthe disclosure and another comprising a reference polypeptide havinglipolytic activity under the same conditions and comparing the results.The improved property may be demonstrated with the methods and assaysdescribed herein. Organoleptic qualities may be evaluated usingprocedures well established in the baking industry, and may include, forexample, the use of a panel of trained taste-testers.

The altered, typically improved, property may be demonstrated in anassay or (bio)chemical analysis.

A variant polypeptide which exhibits a property which is improved inrelation to the reference polypeptide having lipolytic activity is onewhich demonstrates a measurable reduction or increase in the relevantproperty, typically such that the variant is more suited to use as setout herein, for example in a process for the production of a bakedproduct.

The property may thus be decreased by at least 10%, at least 20%, atleast 30%, at least 40% at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95% or at least 99%. Alternatively,the property may be increased by at least 10%, at least 25%, at least50%, at least 100%, at least, 200%, at least 500% or at least 1000%. Thepercentage decrease or increase in this context represents thepercentage decrease or increase in comparison to the referencepolypeptide having lipolytic activity. It is well known to the skilledperson how such percentage changes may be measured—it is a comparison ofthe property of the reference polypeptide having lipolytic activity andthe lipolytic enzyme variant under the same conditions.

The lipolytic enzyme variant according to the disclosure, thecomposition according to the disclosure and/or the pre-mix according tothe disclosure typically result in an improved property in theproduction of a food product or in an improved property of the foodproduct itself. In particular an improved property of a dough comprisingthe variant polypeptide according to the disclosure and/or an improvedproperty of a baked product made using the variant polypeptide accordingto the disclosure.

The term “improved property” herein includes any property of a doughand/or a product obtained from the dough, particularly a baked product,which is improved by the action of the lipolytic enzyme variant, thecomposition according to the disclosure or the pre-mix according to thedisclosure relative to a dough or baked product in which a referencepolypeptide is incorporated.

The improved property may include one or more of, but is not limited to,an increased strength of the dough; an increased elasticity of thedough; increased stability of the dough; an improved extensibility ofthe dough; an increased volume of the baked product; improved flavour ofthe baked product; improved crumb structure of the baked productimproved crispiness; improved oven spring; reduced hardness of a bakedproduct, such as reduced hardness after storage.

The improved property may include a reduced of hardness after storage ofa baked product.

The improved property may be determined by comparison of a dough and/ora baked product prepared with and without addition of the (isolated)polypeptide of the present disclosure in accordance with the methods ofpresent disclosure which are described below in the Examples.Organoleptic qualities may be evaluated using procedures wellestablished in the baking industry, and may include, for example, theuse of a panel of trained testers, e.g. taste-testers, texture-testers.

The term “increased strength of the dough” is defined herein as theproperty of a dough that has generally more elastic properties and/orrequires more work input to mould and shape.

The term “increased elasticity of the dough” is defined herein as theproperty of a dough which has a higher tendency to regain its originalshape after being subjected to a certain physical strain.

The term “increased stability of the dough” is defined herein as theproperty of a dough that is less susceptible to forming faults as aconsequence of mechanical abuse, and thus better at maintaining itsshape and volume and is evaluated by the ratio of height: width of across section of a loaf after normal and/or extended proof.

The term “improved extensibility of the dough” is defined herein as theproperty of a dough that can be subjected to increased strain orstretching without rupture.

An improved dough stability may be an improved shock resistance of adough. Improved shock resistance of a dough may be demonstrated asfollows.

Bread tins filled with well proofed dough (void volume of >70%) undergocontrolled jarring or shocking for example by dropping a tin from setheight, e.g. 9 cm height, just before they enter the oven to be baked.This may be done by simultaneously pulling away 2 blocks having thisdrop-height from below the bottom of the tin. This way the tin dropsover the drop-height and the dough experiences a shock. The shockresistance of a dough is improved if, after baking the dough, the volumeof the loaf is larger as compared to a reference loaf (which may also becalled a control loaf) and/or if the hardness of the loaf after bakingthe dough is lower as compared to a reference loaf (which may also becalled a control).

The term “increased volume of the baked product” is preferably measuredas the volume of a given loaf of bread determined by an automated breadvolume analyser (e.g. BVM-3, TexVol Instruments AB, Viken, Sweden),using ultrasound or laser detection as known in the art. In case thevolume is increased, the property is improved. Alternatively, the heightof the baked product after baking in the same size tin is an indicationof the baked product volume. In case the height of the baked product hasincreased, the volume of the baked product has increased.

The term “improved flavor of the baked product” is evaluated by atrained test panel.

The term “improved crumb structure of the baked product” herein includesthe property of a baked product with finer cells and/or thinner cellwalls in the crumb and/or more uniform/homogenous distribution of gascells in the crumb and is usually evaluated visually by the baker or bydigital image analysis as known in the art (e.g. C-cell, Calibre ControlInternational Ltd, Appleton, Warrington, UK).

The term “improved crispiness” is defined herein as the property of abaked product to give a crispier sensation than a reference product asknown in the art, as well as to maintain this crispier perception for alonger time than a reference product. This property can be quantified bymeasuring a force versus distance curve at a fixed speed in acompression experiment using e.g. a texture analyzer TA-XT Plus (StableMicro Systems Ltd, Surrey, UK), and obtaining physical parameters fromthis compression curve, viz. (i) force of the first peak, (ii) distanceof the first peak, (iii) the initial slope, (iv) the force of thehighest peak, (v) the area under the graph and (vi) the amount offracture events (force drops larger than a certain preset value).Indications of improved crispiness are a higher force of the first peak,a shorter distance of the first peak, a higher initial slope, a higherforce of the highest peak, higher area under the graph and a largernumber of fracture events. A crispier product should score statisticallysignificantly better on at least two of these parameters as compared toa reference product. In the art, “crispiness” is also referred to ascrispness, crunchiness or crustiness, meaning a material with a crispy,crunchy or crusty fracture behaviour.

Oven spring is in bread making defined as the final burst of rising of aloaf, such as a baguette, a batard or a boule, after it is transferredto the oven and before the crust hardens. In some types of bread ovenspring is a desired property and is induced in a defined manner byslashing the dough before it is baked. Slashing is a term that refers tothe process of cutting through the dough skin with a sharp knife.Baguettes are typically made with 3 or 5 diagonal slashes. Oven springmay be assessed visually, for example by an experienced baker judging orranking the oven spring. E.g. Oven spring: 1=incision closed completelyto 5=completely open incision; teared. The oven spring of the bakedproduct may be determined by measuring the crust opening at the largestwidth of the crust opening after baking the dough and cooling the bakedproduct to ambient temperature. An increased oven spring is usuallyconsidered an improved oven spring. For some baked products, an ovenspring having a more rough, jaggered or hairy edge is desirable andconsidered an improved oven spring.

The term “hardness of the baked product” is the opposite of “softness”and is defined herein as the property of a baked product that is lesseasily compressed. Hardness (which may also be referred to as“firmness”) may be evaluated either empirically by a skilled baker ormeasured by the use of a texture analyzer (e.g. TAXT Plus) as known inthe art. The hardness measured within 24 hours after baking is calledinitial hardness. The hardness measured 24 hours or more after baking iscalled hardness after storage, for example after storage for 1, 2, 3, 4or 6 weeks. A reduced hardness may be demonstrated as a reduced initialhardness and/or as a reduced hardness after storage. A reduced hardnessmay be demonstrated by a reduced increase of hardness after storage.

The lipolytic enzyme variant according to the disclosure, thecomposition according to the disclosure and/or the pre-mix according tothe disclosure may result in an improved process for the production adough (e.g. an increased strength of the dough; an increased stabilityof the dough and/or an improved extensibility of the dough) and/or animproved process for the production of a baked product (e.g. anincreased strength of the dough; an increased stability of the doughand/or an improved extensibility of the dough) and/or a baked producthaving at least one improved property.

Typically, the altered properties are determined at ambient conditions.Ambient conditions as used herein include a temperature of 20 to 25degrees C. and a moisture level of 40% humidity. Ambient conditionsherein include a temperature of 20 degrees C. and a moisture level of40% humidity.

The lipolytic enzyme variant according to the disclosure has one or morealtered properties as compared with a reference polypeptide havinglipolytic activity and wherein said variant has at least 70% identitywith the mature polypeptide having lipolytic activity as set out in SEQID NO: 2.

As indicated above the altered, typically improved, property may bedemonstrated in an assay or (bio)chemical analysis. An assay performedat pH 7 or 8 is thought to be more suitable to obtain informationconcerning the preparation of baked products of more alkaline nature,such as cake. An assay performed at pH 5.5 is thought to be moresuitable to obtain information concerning the preparation of bakedproducts of more acidic nature, such as bread.

The altered property may include an altered ratio of the activity onesters of long chain fatty acids: the activity on esters of short chainfatty acids as compared to the reference polypeptide having lipolyticactivity measured under the same conditions.

The altered property may include an increased ratio of the activity onesters of long chain fatty acids: the activity on esters of short chainfatty acids as compared to the reference polypeptide having lipolyticactivity measured under the same conditions.

Ratio of the Activity on Esters of Long Chain Fatty Acids: The Activityon Esters of Short Chain Fatty Acids

In an embodiment of the variant polypeptide according to the disclosurethe altered property includes an increased ratio of the activity onesters of long chain fatty acids: the activity on esters of short chainfatty acids as compared to the reference polypeptide having lipolyticactivity measured under the same conditions.

The disclosure provides lipolytic enzyme variants having an alteredratio of the activity on esters of long chain fatty acids: the activityon esters of short chain fatty acids as compared to the referencepolypeptide having lipolytic activity measured under the sameconditions.

In an embodiment, the variant polypeptide according to the disclosurehas an amino acid sequence which, when aligned with the amino acidsequence as set out in SEQ ID NO: 2, comprises at least one substitutionof an amino acid residue at a position corresponding to any of thepositions 53, 112, 141, 178, 179, 182, 202, 203, 235, 282, 284,

said positions being defined with reference to SEQ ID NO: 2, and whereinthe variant has one or more altered properties as compared with areference polypeptide having lipolytic activity and wherein said varianthas at least 70% identity with the mature polypeptide having lipolyticactivity as set out in SEQ ID NO: 2.

In an embodiment of the variant polypeptide according to the disclosure,the reference polypeptide comprises an amino acid sequence as set out inamino acids 34 to 304 of SEQ ID NO: 2 and the mature polypeptidecomprises an amino acid sequence as set out in amino acids 34 to 304 ofSEQ ID NO: 2.

In an embodiment, the variant polypeptide according to the disclosurehas an amino acid sequence which, when aligned with the amino acidsequence as set out in SEQ ID NO: 2, comprises one or more of the aminoacid substitutions selected from

-   -   Y53S    -   S112T    -   F141M    -   A178G    -   V179L    -   V179M    -   L182F    -   P202A    -   R203M    -   P235L    -   L282F    -   I284Q        said position being defined with reference to SEQ ID NO: 2.

The ratio of the activity on esters of long chain fatty acids: theactivity on esters of short chain fatty acids is determined at a certainpH such as pH 5.5 or pH 7 or pH 8.

The determination of the activity on esters of long chain fatty acidsmay be done via measuring using suitable assays at pH 5.5 such as Assay6A, using pNP-stearate as substrate.

The determination of the activity on esters of long chain fatty acidsmay be done via measuring using suitable assays at pH 7 such as Assay6B, using pNP-stearate as substrate.

The determination of the activity on esters of long chain fatty acidsmay be done via measuring using suitable assays at pH 8 such asanalogous to Assay 6A and using a pH 8 buffer.

The determination of the activity on esters of long chain fatty acids atmay be done via measuring using suitable assays at pH 5.5 such as Assay5A, using pNP-oleate as substrate.

The determination of the activity on esters of long chain fatty acidsmay be done via measuring using suitable assays at pH 7 such as Assay5B, using pNP-oleate as substrate.

The determination of the activity on esters of long chain fatty acidsmay be done via measuring using suitable assays at pH 5.5 such as Assay4A, using pNP-lineolate as substrate.

The determination of the activity on esters of long chain fatty acidsmay be done via measuring using suitable assays at pH 7 such as Assay4B, using pNP-lineolate as substrate.

The determination of the activity on esters of short chain fatty acidsmay be done via measuring using suitable assays at pH 5.5 such as Assay7A, using pNP-butyrate as substrate.

The determination of the activity on esters of short chain fatty acidsmay be done via measuring using suitable assays at pH 7 such as Assay7B, using pNP-butyrate as substrate.

The determination of the activity on esters of short chain fatty acidsmay be done via measuring using suitable assays at pH 8 such asanalogous to Assay 7A and using a pH 8 buffer.

The ratio of the activity on esters of long chain fatty acids: theactivity on esters of short chain fatty acids may be determined viameasuring the particular activities using suitable assays as describedherein and calculating the ratio analogous to “Exemplary determinationand calculation of galactolipase to TAG-lipase activity ratio of variant#” in the Materials and Methods herein.

For example the ratio of the activity on esters of long chain fattyacids: the activity on esters of short chain fatty acids at pH 5.5 maybe determined by applying suitable assays at pH 5.5 as described herein(e.g. applying Assay 5A, Assay 7A) and calculating the ratio analogousto “Exemplary determination and calculation of galactolipase toTAG-lipase activity ratio of variant #” in the Materials and Methodsherein.

More examples are given in the Example section herein. See“Determination of altered properties” and Tables 3 to 7 thereafter.

In an aspect of the disclosure the altered ratio of the activity onesters of long chain fatty acids: the activity on esters of short chainfatty acids as compared to the reference polypeptide having lipolyticactivity measured under the same conditions is an increased ratio of theactivity on esters of long chain fatty acids: the activity on esters ofshort chain fatty acids as compared to the reference polypeptide havinglipolytic activity measured under the same conditions.

In an aspect of the disclosure the increased ratio of the activity onesters of long chain fatty acids: the activity on esters of short chainfatty acids, as compared to the reference polypeptide having lipolyticactivity measured under the same conditions, the altered ratio of theactivity on esters of long chain fatty acids: the activity on esters ofshort chain fatty acids is an increased ratio of the activity on estersof C12-C18 fatty acid: the activity on esters of C4-C8 fatty acids ascompared to the reference polypeptide having lipolytic activity (such asthe polypeptide of SEQ ID NO: 2) measured under the same conditions.

In an aspect of the disclosure the increased ratio of the activity onesters of long chain fatty acids: the activity on esters of short chainfatty acids, as compared to the reference polypeptide having lipolyticactivity measured under the same conditions, the increased ratio of theactivity on esters of long chain fatty acids: the activity on esters ofshort chain fatty acids is an increased ratio of the activity on estersof C16-C18 fatty acids: the activity on esters of C4-C6 fatty acids ascompared to the reference polypeptide having lipolytic activity measuredunder the same conditions.

In an aspect of the disclosure the increased ratio of the activity onesters of long chain fatty acids: the activity on esters of short chainfatty acids, as compared to the reference polypeptide having lipolyticactivity measured under the same conditions, the increased ratio of theactivity on esters of long chain fatty acids: the activity on esters ofshort chain fatty acids is an increased ratio of the activity on estersof C18 fatty acids: the activity on esters of C4 fatty acids as comparedto the reference polypeptide having lipolytic activity measured underthe same conditions.

In an aspect of the disclosure the increased ratio of the activity onesters of long chain fatty acids: the activity on esters of short chainfatty acids, as compared to the reference polypeptide having lipolyticactivity measured under the same conditions, the increased ratio of theactivity on esters of long chain fatty acids: the activity on esters ofshort chain fatty acids is an increased ratio of the activity on estersof C18:2 fatty acids: the activity on esters of C4 fatty acids ascompared to the reference polypeptide having lipolytic activity measuredunder the same conditions.

In an aspect of the disclosure the increased ratio of the activity onesters of short chain fatty acids: the activity on esters of long chainfatty acids is an increased ratio of the activity on pNP-stearate:theactivity on pNP-butyrate as compared to the reference polypeptide havinglipolytic activity measured under the same conditions.

The increased ratio of the activity on pNP-stearate: the activity onpNP-butyrate can be determined at pH 5.5.

The increased ratio of the activity on pNP-stearate: the activity onpNP-butyrate can be determined at pH 7.

The increased ratio of the activity on esters of long chain fatty acids:the activity on esters of short chain fatty acids can be an increasedratio of the activity on pNP-oleate: the activity on pNP-butyrate ascompared to the reference polypeptide having lipolytic measured underthe same conditions.

The increased ratio of the activity on pNP-oleate: the activity onpNP-butyrate can be determined at pH 5.5.

The increased ratio of the activity on pNP-oleate: the activity onpNP-butyrate can be determined at pH 7.

The increased ratio of the activity on esters of long chain fatty acids:the activity on esters of short chain fatty acids can be an increasedratio of the activity on Lineolate: the activity on pNP-butyrate ascompared to the reference polypeptide having lipolytic activity measuredunder the same conditions.

The increased ratio of the activity on pNP-lineolate: the activity onpNP-butyrate can be determined at pH 5.5. (E.g. Assays 4A and 7A andcalculating the ratio as described herein)

The increased ratio of the activity on pNP-lineolate: the activity onpNP-butyrate can be determined at pH 7. (E.g. Assays 4B and 7B andcalculating the ratio as described herein)

An increased ratio of the activity on esters of long chain fatty acids:the activity on esters of short chain fatty acids as compared to thereference polypeptide having lipolytic activity measured under the sameconditions may for example indicate that the variant is more suitablethan the reference polypeptide for bakery applications made using fat(typically shortening or butter) for example pastry products. A typicalexample being Danish pastry and croissants.

The lipolytic enzyme variant according to the disclosure may be used toavoid flavour defects associated with short chain fatty acids forexample in cake and/pastry products such as croissants.

The disclosure provides a nucleic acid sequence encoding the variantpolypeptide according to the disclosure, i.e. provides a nucleic acidsequence encoding the lipolytic enzyme variant of the disclosure.

The disclosure further provides a nucleic acid sequence encoding alipolytic variant which comprises a sequence that has at least 70%sequence identity to the mature polypeptide having lipolytic activity asset out in SEQ ID NO: 2.

A nucleic acid sequence of the disclosure may comprise a polynucleotidesequence encoding a variant polypeptide of the disclosure which has atleast 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to the mature polypeptide having lipolytic activity as set outin SEQ ID NO: 2.

In an aspect the nucleic acid sequence of the disclosure comprises apolynucleotide sequence encoding a variant polypeptide of the disclosurewhich has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to the mature polypeptide having lipolytic activity asset out in amino acids 34 to 304 of SEQ ID NO: 2.

The disclosure provides a nucleic acid construct comprising the nucleicacid sequence of the disclosure operably linked to one or more controlsequences capable of directing the expression of a lipolytic enzyme in asuitable expression host.

The disclosure provides a recombinant host cell comprising a recombinantexpression vector comprising the nucleic acid construct of thedisclosure.

The disclosure provides a method for producing a lipolytic polypeptidevariant according to the disclosure comprising cultivating the host cellof the disclosure under conditions conducive to production of thelipolytic enzyme variant and recovering the lipolytic enzyme variant.

The method to produce the lipolytic polypeptide variant according tothis disclosure includes a method to produce all variants describedherein.

The term “complementary strand” can be used interchangeably with theterm “complement”. The complementary strand of a nucleic acid can be thecomplement of a coding strand or the complement of a non-coding strand.When referring to double-stranded nucleic acids, the complement of anucleic acid encoding a polypeptide refers to the complementary strandof the strand encoding the amino acid sequence or to any nucleic acidmolecule containing the same. Typically, the reverse complementarystrand is intended.

The term “control sequence” can be used interchangeably with the term“expression-regulating nucleic acid sequence”. The term as used hereinrefers to nucleic acid sequences necessary for and/or affecting theexpression of an operably linked coding sequence in a particular hostorganism or in vitro. When two nucleic acid sequences are operablylinked, they usually will be in the same orientation and also in thesame reading frame. They usually will be essentially contiguous,although this may not be required. The expression-regulating nucleicacid sequences, such as inter alia appropriate transcription initiation,termination, promoter, leader, signal peptide, propeptide,prepropeptide, or enhancer sequences; Shine-Dalgarno sequence, repressoror activator sequences; efficient RNA processing signals such assplicing and polyadenylation signals; sequences that stabilizecytoplasmic mRNA; sequences that enhance translation efficiency (e.g.,ribosome binding sites); sequences that enhance protein stability; andwhen desired, sequences that enhance protein secretion, can be anynucleic acid sequence showing activity in the host organism of choiceand can be derived from genes encoding proteins, which are eitherendogenous or heterologous to a host cell. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.When desired, the control sequence may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleic acidsequence encoding a polypeptide. Control sequences may be optimized totheir specific purpose.

The term “derived from” also includes the terms “originated from,”“obtained from,” “obtainable from,” “isolated from,” and “created from,”and generally indicates that one specified material find its origin inanother specified material or has features that can be described withreference to another specified material. As used herein, a substance(e.g., a nucleic acid molecule or polypeptide) “derived from” amicroorganism preferably means that the substance is native to thatmicroorganism.

As used herein, the term “endogenous” refers to a nucleic acid or aminoacid sequence naturally occurring in a host cell.

The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription, posttranscriptional modification, translation, post-translationalmodification, and secretion.

An “expression vector” comprises a polynucleotide coding for apolypeptide, operably linked to the appropriate control sequences (suchas a promoter, and transcriptional and translational stop signals) forexpression and/or translation in vitro, or in the host cell of thepolynucleotide.

The expression vector may be any vector (e.g., a plasmid or virus),which can be conveniently subjected to recombinant DNA procedures andcan bring about the expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thecell into which the vector is to be introduced. The vectors may belinear or closed circular plasmids. The vector may be an autonomouslyreplicating vector, i.e. a vector, which exists as an extra-chromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a plasmid, an extra-chromosomal element, amini-chromosome, or an artificial chromosome. Alternatively, the vectormay be one which, when introduced into the host cell, is integrated intothe genome and replicated together with the chromosome(s) into which ithas been integrated. The integrative cloning vector may integrate atrandom or at a predetermined target locus in the chromosomes of the hostcell. The vector system may be a single vector or plasmid or two or morevectors or plasmids, which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon.

A “host cell” as defined herein is an organism suitable for geneticmanipulation and one which may be cultured at cell densities useful forindustrial production of a target product, such as a polypeptideaccording to the present disclosure. A host cell may be a host cellfound in nature or a host cell derived from a parent host cell aftergenetic manipulation or classical mutagenesis. Advantageously, a hostcell is a recombinant host cell.

A host cell may be a prokaryotic, archaebacterial or eukaryotic hostcell. A prokaryotic host cell may be, but is not limited to, a bacterialhost cell. A eukaryotic host cell may be, but is not limited to, ayeast, a fungus, an amoeba, an alga, a plant, an animal cell, such as amammalian or an insect cell.

The term “heterologous” as used herein refers to nucleic acid or aminoacid sequences not naturally occurring in a host cell. In other words,the nucleic acid or amino acid sequence is not identical to thatnaturally found in the host cell.

The term “hybridization” means the pairing of substantiallycomplementary strands of oligomeric compounds, such as nucleic acidcompounds.

Hybridization may be performed under low, medium or high stringencyconditions. Low stringency hybridization conditions comprise hybridizingin 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed bytwo washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature ofthe washes can be increased to 55° C. for low stringency conditions).Medium stringency hybridization conditions comprise hybridizing in 6×SSCat about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at60° C., and high stringency hybridization conditions comprisehybridizing in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.

A nucleic acid or polynucleotide sequence is defined herein as anucleotide polymer comprising at least 5 nucleotide or nucleic acidunits. A nucleotide or nucleic acid refers to RNA and DNA. The terms“nucleic acid” and “polynucleotide sequence” are used interchangeablyherein.

A “peptide” refers to a short chain of amino acid residues linked bypeptide (amide) bonds. The shortest peptide, a dipeptide, consists of 2amino acids joined by single peptide bond.

The term “polypeptide” refers to a molecule comprising amino acidresidues linked by peptide bonds and containing more than five aminoacid residues. The term “protein” as used herein is synonymous with theterm “polypeptide” and may also refer to two or more polypeptides. Thus,the terms “protein” and “polypeptide” can be used interchangeably.Polypeptides may optionally be modified (e.g., glycosylated,phosphorylated, acylated, farnesylated, prenylated, sulfonated, and thelike) to add functionality. Polypeptides exhibiting activity in thepresence of a specific substrate under certain conditions may bereferred to as enzymes. It will be understood that, as a result of thedegeneracy of the genetic code, a multitude of nucleotide sequencesencoding a given polypeptide may be produced. An “enzyme” is apolypeptide that catalyzes a chemical reaction.

An “isolated nucleic acid fragment” is a nucleic acid fragment that isnot naturally occurring as a fragment and would not be found in thenatural state.

The term “isolated polypeptide” as used herein means a polypeptide thatis removed from at least one component, e.g. other polypeptide material,with which it is naturally associated. The isolated polypeptide may befree of any other impurities. The isolated polypeptide may be at least50% pure, e.g., at least 60% pure, at least 70% pure, at least 75% pure,at least 80% pure, at least 85% pure, at least 90% pure, or at least 95%pure, 96%, 97%, 98%, 99%, 99.5%, 99.9% as determined by SDS-PAGE or anyother analytical method suitable for this purpose and known to theperson skilled in the art. An isolated polypeptide may be produced by arecombinant host cell.

The lipolytic enzyme variant according to the disclosure can berecovered and purified from recombinant cell cultures by methods knownin the art (Protein Purification Protocols, Methods in Molecular Biologyseries by Paul Cutler, Humana Press, 2004).

The lipolytic enzyme variant includes naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the polypeptides of the present disclosure may be glycosylated or may benon-glycosylated. In addition, polypeptides of the disclosure may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

In the disclosure, a lipolytic polypeptide variant may be provided inthe form of pre-polypeptide variant or (mature) polypeptide variant. Acorresponding nucleic acid sequence may also be provided, i.e. apolynucleotide that encodes a pre-lipolytic polypeptide variant or a(mature) lipolytic polypeptide variant may be provided.

The term “nucleic acid construct” is herein referred to as a nucleicacid molecule, either single- or double-stranded, which is isolated froma naturally occurring gene or which has been modified to containsegments of nucleic acid which are combined and juxtaposed in a mannerwhich would not otherwise exist in nature. The term nucleic acidconstruct is synonymous with the term “expression cassette” when thenucleic acid construct contains all the control sequences required forexpression of a coding sequence, wherein said control sequences areoperably linked to said coding sequence.

The term “promoter” is defined herein as a DNA sequence that is bound byRNA polymerase and directs the polymerase to the correct downstreamtranscriptional start site of a nucleic acid sequence to initiatetranscription. A promoter may also comprise binding sites forregulators.

The term “recombinant” when used in reference to a cell, nucleic acid,protein or vector, indicates that the cell, nucleic acid, protein orvector, has been modified by the introduction of a heterologous nucleicacid or protein or the alteration of a native nucleic acid or protein,or that the cell is derived from a cell so modified. Thus, for example,recombinant cells express genes that are not found within the native(non-recombinant) form of the cell or express native genes that areotherwise abnormally expressed, underexpressed or not expressed at all.The term “recombinant” is synonymous with “genetically modified” and“transgenic”.

The terms “sequence identity” or “sequence homology” are usedinterchangeably herein. For the purpose of this disclosure, it isdefined here that in order to determine the percentage of sequencehomology or sequence identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes. In order to optimize the alignment between the two sequencesgaps may be introduced in any of the two sequences that are compared.Such alignment can be carried out over the full length of the sequencesbeing compared. Alternatively, the alignment may be carried out over ashorter length, for example over about 20, about 50, about 100 or morenucleotides/bases or amino acids. The sequence identity is thepercentage of identical matches between the two sequences over thereported aligned region.

A comparison of sequences and determination of percentage of sequenceidentity between two sequences can be accomplished using a mathematicalalgorithm. The skilled person will be aware of the fact that severaldifferent computer programs are available to align two sequences anddetermine the identity between two sequences (Kruskal, J. B. (1983) Anoverview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.),Time warps, string edits and macromolecules: the theory and practice ofsequence comparison, pp. 1-44 Addison Wesley). The percent sequenceidentity between two amino acid sequences or between two nucleotidesequences may be determined using the Needleman and Wunsch algorithm forthe alignment of two sequences. (Needleman, S. B. and Wunsch, C. D.(1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences andnucleotide sequences can be aligned by the algorithm. TheNeedleman-Wunsch algorithm has been implemented in the computer programNEEDLE. For the purpose of this disclosure the NEEDLE program from theEMBOSS package was used (version 2.8.0 or higher, EMBOSS: The EuropeanMolecular Biology Open Software Suite (2000) Rice, P. Longden, I. andBleasby, A. Trends in Genetics 16, (6) pp 276-277,http://emboss.bioinformatics.nl/). For protein sequences EBLOSUM62 isused for the substitution matrix. For nucleotide sequence, EDNAFULL isused. The optional parameters used are a gap-open penalty of 10 and agap extension penalty of 0.5. The skilled person will appreciate thatall these different parameters will yield slightly different results butthat the overall percentage identity of two sequences is notsignificantly altered when using different algorithms.

After alignment by the program NEEDLE as described above the percentageof sequence identity between a query sequence and a sequence of thedisclosure is calculated as follows: Number of corresponding positionsin the alignment showing an identical amino acid or identical nucleotidein both sequences divided by the total length of the alignment aftersubtraction of the total number of gaps in the alignment. The identitydefined as herein can be obtained from NEEDLE by using the NOBRIEFoption and is labelled in the output of the program as“longest-identity”.

The nucleic acid and protein sequences of the present disclosure canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the disclosure. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the disclosure. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See the homepage of the NationalCenter for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.

As indicated above the variant polypeptide according to the disclosuremay be an isolated, substantially pure, pure, recombinant or syntheticpolypeptide.

In an embodiment, the variant polypeptide according to the disclosure isa non-naturally occurring polypeptide.

A “pure enzyme” is synonymous to “pure polypeptide” and means apolypeptide that is removed from at least one component, e.g. otherpolypeptide material, with which it is naturally associated. Thepolypeptide may be free of any other impurities. The polypeptide may beat least 50% pure, e.g., at least 60% pure, at least 70% pure, at least75% pure, at least 80% pure, at least 85% pure, at least 90% pure, or atleast 95% pure, 96%, 97%, 98%, 99%, 99.5%, 99.9% as determined bySDS-PAGE or any other analytical method suitable for this purpose andknown to the person skilled in the art. An isolated polypeptide may beproduced by a recombinant host cell.

The term “substantially pure” with regard to polypeptides refers to apolypeptide preparation which contains at the most 50% by weight ofother polypeptide material. The polypeptides disclosed herein arepreferably in a substantially pure form. In particular, it is preferredthat the polypeptides disclosed herein are in “essentially pure form”,i.e. that the polypeptide preparation is essentially free of otherpolypeptide material. Optionally, the polypeptide may also beessentially free of non-polypeptide material such as nucleic acids,lipids, media components, and the like. Herein, the term “substantiallypure polypeptide” is synonymous with the terms “isolated polypeptide”and “polypeptide in isolated form”. The term “substantially pure” withregard to polynucleotide refers to a polynucleotide preparation whichcontains at the most 50% by weight of other polynucleotide material. Thepolynucleotides disclosed herein are preferably in a substantially pureform. In particular, it is preferred that the polynucleotide disclosedherein are in “essentially pure form”, i.e. that the polynucleotidepreparation is essentially free of other polynucleotide material.Optionally, the polynucleotide may also be essentially free ofnon-polynucleotide material such as polypeptides, lipids, mediacomponents, and the like. Herein, the term “substantially purepolynucleotide” is synonymous with the terms “isolated polynucleotide”and “polynucleotide in isolated form”.

A “synthetic molecule”, such as a synthetic nucleic acid or a syntheticpolypeptide is produced by in vitro chemical or enzymatic synthesis. Itincludes, but is not limited to, variant nucleic acids made with optimalcodon usage for host organisms of choice.

A synthetic nucleic acid may be optimized for codon use, preferablyaccording to the methods described in WO2006/077258 and/or WO2008000632,which are herein incorporated by reference. WO2008/000632 addressescodon-pair optimization. Codon-pair optimization is a method wherein thenucleotide sequences encoding a polypeptide that have been modified withrespect to their codon-usage, in particular the codon-pairs that areused, are optimized to obtain improved expression of the nucleotidesequence encoding the polypeptide and/or improved production of theencoded polypeptide. Codon pairs are defined as a set of two subsequenttriplets (codons) in a coding sequence. Those skilled in the art willknow that the codon usage needs to be adapted depending on the hostspecies, possibly resulting in variants with significant homologydeviation from SEQ ID NO: 1, but still encoding the polypeptideaccording to the disclosure.

As used herein, the terms “variant”, “derivative”, “mutant” or“homologue” can be used interchangeably. They can refer to eitherpolypeptides or nucleic acids. Variants include substitutions,insertions, deletions, truncations, transversions, and/or inversions, atone or more locations relative to a reference sequence. Variants can bemade for example by site-saturation mutagenesis, scanning mutagenesis,insertional mutagenesis, random mutagenesis, site-directed mutagenesis,and directed-evolution, as well as various other recombinationapproaches known to a skilled person in the art. Variant genes ofnucleic acids may be synthesized artificially by known techniques in theart.

A polypeptide according to the present disclosure may be encoded by anysuitable polynucleotide sequence. Typically, a polynucleotide sequenceis codon optimized, or a codon pair optimized sequence for expression ofa polypeptide as disclosed herein in a particular host cell. Thepolynucleotides according to the disclosure may be optimized in theircodon use, preferably according to the methods described inWO2006/077258 and/or WO2008/000632. WO2008/000632 addresses codon-pairoptimization. Codon-pair optimisation is a method wherein the nucleotidesequences encoding a polypeptide are modified with respect to theircodon-usage, in particular the codon-pairs that are used, to obtainimproved expression of the nucleotide sequence encoding the polypeptideand/or improved production of the encoded polypeptide. Codon pairs aredefined as a set of two subsequent triplets (codons) in a codingsequence.

A polypeptide of the disclosure may be encoded by a polynucleotidesequence that comprises appropriate control sequences and/or signalsequences, for example for secretion.

A polypeptide of the disclosure may be encoded by a polynucleotide thathybridizes under medium stringency, preferably under high stringencyconditions to the complementary strand of the mature polypeptide codingsequence of SEQ ID NO: 2 (or the corresponding wild-type sequence or asequence codon optimized or codon pair optimized for expression in aheterologous organism, such as a Bacillus, for example Bacillussubtilis).

A polypeptide of the disclosure may also be encoded by a nucleic acidthat has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identity to a mature polypeptide coding sequence of SEQ IDNO: 2 (or the corresponding wild-type sequence or a sequence codonoptimized or codon pair optimized for expression in a heterologousorganism, such as a Bacillus, for example Bacillus subtilis).

A polypeptide of the disclosure may also be a variant of a maturepolypeptide of SEQ ID NO: 1, comprising a substitution, deletion and/orinsertion at one or more positions of the mature polypeptide SEQ IDNO: 1. A variant of the mature polypeptide of SEQ ID NO: 1 may be anamino acid sequence that differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or12 amino acids from the amino acids of the mature polypeptide of SEQ IDNO: 1.

In one embodiment, the present disclosure features a biologically activefragment of a polypeptide as disclosed herein.

Biologically active fragments of a polypeptide of the disclosure includepolypeptides comprising amino acid sequences sufficiently identical toor derived from the amino acid sequence of the lipolytic enzyme variant,which include fewer amino acids than the full-length protein but whichexhibits at least one biological activity of the correspondingfull-length protein. Typically, biologically active fragments comprise adomain or motif with at least one activity of the lipolytic enzymevariant. A biologically active fragment may for instance comprise acatalytic domain. A biologically active fragment of a protein of thedisclosure can be a polypeptide which is, for example, 10, 25, 50, 100or more amino acids in length. Moreover, other biologically activeportions, in which other regions of the protein are deleted, can beprepared by recombinant techniques and evaluated for one or more of thebiological activities of the native form of a polypeptide of thedisclosure.

The disclosure also features nucleic acid fragments which encode theabove biologically active fragments of the lipolytic enzyme variant.

A polypeptide according to the present disclosure may be a fusionprotein. Techniques for producing fusion polypeptides are known in theart, and include ligating the coding sequences encoding the polypeptidesso that they are in frame. Expression of the fused polypeptide is undercontrol of the same promoter(s) and terminator. The hybrid polypeptidesmay comprise a combination of partial or complete polypeptide sequencesobtained from at least two different polypeptides wherein one or moremay be heterologous to a host cell. Such fusion polypeptides from atleast two different polypeptides may comprise a binding domain from onepolypeptide, operably linked to a catalytic domain from a secondpolypeptide. Examples of fusion polypeptides and signal sequence fusionsare for example as described in WO2010/121933, WO2013/007820 andWO2013/007821.

A polypeptide of the disclosure may be a naturally occurring polypeptideor a genetically modified or recombinant polypeptide.

A polypeptide of the disclosure may be purified. Purification ofproteins is known to a skilled person in the art.

The term “enzymatic activity”, sometimes also referred to as “catalyticactivity” or “catalytic efficiency”, is generally known to the personskilled in the art and refers to the conversion rate of an enzyme and isusually expressed by means of the ratio k_(kat)/K_(M), wherein k_(kat)is the catalytic constant (also referred to as turnover number) and theK_(M) value corresponds to the substrate concentration, at which thereaction rate lies at half its maximum value. Alternatively, theenzymatic activity of an enzyme can also be specified by the specificactivity (μmol of converted substrate×mg⁻¹×min⁻¹; cf. above) or thevolumetric activity (μmol of converted substrate×mL⁻¹×min⁻¹; cf. above).Reference can also be made to the general literature such as Structureand Mechanism in Protein Science: A guide to enzyme catalysis andprotein folding, Alan Fersht, W. H. Freeman, 1999; Fundamentals ofEnzyme Kinetics, Athel Cornish-Bowden, Wiley-Blackwell 2012 and Voet etal., “Biochemie” [Biochemistry], 1992, VCH-Verlag, Chapter 13, pages331-332 with respect to enzymatic activity.

There are several ways of inserting a nucleic acid sequence into anucleic acid construct or an expression vector which are known to askilled person in the art, see for instance Sambrook & Russell,Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold SpringHarbor, N.Y., 2001. It may be desirable to manipulate a nucleic acidsequence encoding a polypeptide of the present disclosure with controlsequences, such as promoter and terminator sequences.

A promoter may be any appropriate promoter sequence suitable for aeukaryotic or prokaryotic host cell, which shows transcriptionalactivity, including mutant, truncated, and hybrid promoters, and may beobtained from polynucleotides encoding extracellular or intracellularpolypeptides either endogenous (native) or heterologous (foreign) to thecell. The promoter may be a constitutive or inducible promoter. Aninducible promoter may be, for example, a starch inducible promoter.

Suitable promoters will be known to the skilled person. In a specificembodiment, promoters are preferred that are capable of directing a highexpression level of the polypeptides according to the disclosure in afungus or yeast. Such promoters are known in the art.

A variety of promoters can be used that are capable of directingtranscription in the host cells of the disclosure. Preferably thepromoter sequence is derived from a highly expressed gene. Strongconstitutive promoters are well known and an appropriate one may beselected according to the specific sequence to be controlled in the hostcell. Examples of preferred highly expressed genes from which promotersare preferably derived and/or which are comprised in preferredpredetermined target loci for integration of expression constructs.Examples of suitable promotors are listed in WO 2009/106575, includingexamples of suitable promotors in filamentous fungi. All of thepromoters mentioned therein are readily available in the art.

Any terminator which is functional in a cell as disclosed herein may beused, which are known to a skilled person in the art. Examples ofsuitable terminator sequences in filamentous fungi include terminatorsequences of a filamentous fungal gene, for example those listed in WO2009/106575.

The disclosure also relates to a vector which comprises a nucleic acidof the disclosure, said vector comprises at least an autonomousreplication sequence and a nucleic acid as described herein.

The vector may be any vector (e.g. a plasmid or a virus), which can beconveniently subjected to recombinant DNA procedures. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. Preferably, thevector is a plasmid. The vector may be a linear or a closed circularplasmid. The vector may further comprise a, preferably non-selective,marker that allows for easy determination of the vector in the hostcell. Suitable markers include GFP and DsRed. The chance of geneconversion or integration of the vector into the host genome ispreferably minimized. The vector according to the disclosure may be anextra-chromosomal vector. Such a vector preferably lacks significantregions of homology with the genome of the host to minimize the chanceof integration into the host genome by homologous recombination. Theperson skilled in the art knows how to construct a vector with minimalchance of integration into the genome. This may be achieved by usingcontrol sequences, such as promoters and terminators, which originatefrom another species than the host species. Other ways of reducinghomology are by modifying codon usage and introduction of silentmutations. The person skilled in the art knows that the type of hostcell, the length of the regions of homology to the host cell genomepresent in the vector, and the percentage of homology between saidregions of homology in the vector and the host chromosome will determinewhether and in which amount the vector will integrate into the host cellgenome.

The autonomous replication sequence may be any suitable sequenceavailable to the person skilled in the art that allows for plasmidreplication that is independent of chromosomal replication.

The origin of replication may be any plasmid replicator mediatingautonomous replication that functions in a cell. The term “origin ofreplication” or “plasmid replicator” is defined herein as a nucleotidesequence that enables a plasmid or vector to replicate in vivo. Examplesof bacterial origins of replication are the origins of replication ofplasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication inE. coli, RSF1010 permitting replication in Pseudomonas is described,e.g., by F. Heffron et al., in Proc. Nat'l Acad. Sci. USA 72(9):3623-27(September 1975), and pUB110, pE194, pTA1060, and pAMß1 permittingreplication in Bacillus.

Preferably, the autonomous replication sequence used in filamentousfungi is the AMA1 replicon (Gems et al., 1991 Gene. 98(1):61-7).Telomeric repeats may also result in autonomous replication (In vivolinearization and autonomous replication of plasmids containing humantelomeric DNA in Aspergillus nidulans, Aleksenko et al. Molecular andGeneral Genetics MGG, 1998—Volume 260, Numbers 2-3, 159-164, DOI:10.1007/s004380050881). CEN/ARS sequences and 3p vector sequences fromyeast may also be suitable.

A vector or expression construct for a given host cell may thus comprisethe following elements operably linked to each other in a consecutiveorder from the 5′-end to 3′-end relative to the coding strand of thesequence encoding the compound of interest or encoding a compoundinvolved in the synthesis of the compound of interest: (1) a promotersequence capable of directing transcription of a nucleic acid of thedisclosure; (2) optionally a sequence to facilitate the translation ofthe transcribed RNA, for example a ribosome binding site (also indicatedas Shine Delgarno sequence) in prokaryotes, or a Kozak sequence ineukaryotes (3) optionally, a signal sequence capable of directingsecretion of the lipolytic enzyme variant encoded by the nucleic acid ofthe disclosure from the given host cell into a culture medium; (4) anucleic acid of the disclosure, as described herein; and preferably also(5) a transcription termination region (terminator) capable ofterminating transcription downstream of the nucleic acid of thedisclosure. The vector may comprise these and/or other control sequencesas described herein.

Downstream of a nucleic acid of the disclosure there may be a3′-untranslated region containing one or more transcription terminationsites (e. g. a terminator, herein also referred to as a stop codon). Theorigin of the terminator is not critical. The terminator can, forexample, be native to the DNA sequence encoding the polypeptide.However, preferably a bacterial terminator is used in bacterial hostcells and a filamentous fungal terminator is used in filamentous fungalhost cells. More preferably, the terminator is endogenous to the hostcell (in which the nucleotide sequence encoding the polypeptide is to beexpressed). In the transcribed region, a ribosome binding site fortranslation may be present. The coding portion of the mature transcriptsexpressed by the constructs will include a start codon, usually AUG (orATG), but there are also alternative start codons, such as for exampleGUG (or GTG) and UUG (or TTG), which are used in prokaryotes. Also astop or translation termination codon is appropriately positioned at theend of the polypeptide to be translated.

Enhanced expression of a lipolytic enzyme variant of the disclosure mayalso be achieved by the selection of homologous and heterologousregulatory regions, e. g. promoter, secretion leader and/or terminatorregions, which may serve to increase expression and, if desired,secretion levels of the protein of interest from the expression hostand/or to provide for the inducible control of the expression of acompound of interest or a compound involved in the synthesis of acompound of interest.

The vector comprising at least an autonomous replication sequence and anucleic acid of the disclosure, also referred to herein as “vector (orexpression vector) of the disclosure” can be designed for expression ofthe nucleic acid in a prokaryotic or a eukaryotic cell. For example, alipolytic enzyme variant of the disclosure can be produced in bacterialcells such as E. coli or Bacilli, insect cells (using baculovirusexpression vectors), fungal cells, such as yeast cells, or mammaliancells. Suitable host cells are discussed herein and further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

In order to identify and select cells which harbour a nucleic acidand/or vector of the disclosure, a gene that encodes a selectable marker(e.g., resistance to antibiotics) is optionally introduced into thevector and/or host cells along with the nucleic acid of the disclosure.Preferred selectable markers include, but are not limited to those whichconfer resistance to drugs or which complement a defect in the hostcell. The person skilled in the art knows how choose and apply suchmarkers. Examples of suitable markers are listed in WO 2009/106575.

Expression of proteins in prokaryotes is often carried out with vectorscontaining constitutive or inducible promoters directing the expressionof either fusion or non-fusion proteins. Fusion vectors add a number ofamino acids to a protein encoded therein, e.g. to the amino terminus ofthe recombinant protein. Such fusion vectors typically serve threepurposes: 1) to increase expression of recombinant protein; 2) toincrease the solubility of the recombinant protein; and 3) to aid in thepurification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.

The present disclosure also provides a host cell comprising a nucleicacid or an expression vector as disclosed herein. A suitable host cellmay be a mammalian, insect, plant, fungal, or algal cell, or a bacterialcell.

The host cell may be a prokaryotic cell. In an aspect, the prokaryotichost cell is a bacterial cell. Examples of bacterial cells are listed inWO 2009/106575.

According to an embodiment, the host cell according to the disclosure isa eukaryotic host cell. Preferably, the eukaryotic cell is a mammalian,insect, plant, fungal, or algal cell. Examples of eukaryotic host cellare listed in WO 2009/106575.

The eukaryotic cell may be a fungal cell, for example a yeast cell, suchas a cell of the genus Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia. More specifically, ayeast cell may be from Kluyveromyces lactis, Saccharomyces cerevisiae,Hansenula polymorpha, Yarrowia lipolytica and Pichia pastoris, Candidakrusei.

Preferred filamentous fungal cells belong to a species of an Acremonium,Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Talaromyces,Rasamsonia, Thielavia, Fusarium or Trichoderma genus, and mostpreferably a species of Aspergillus niger, Acremonium alabamense,Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae,Aspergillus fumigatus, Talaromyces emersonii, Rasamsonia emersonii,Aspergillus oryzae, Chrysosporium lucknowense, Fusarium oxysporum,Myceliophthora thermophila, Trichoderma reesei, Thielavia terrestris orPenicillium chrysogenum. A more preferred filamentous fungal host cellbelongs to the genus Aspergillus, more preferably the host cell belongsto the species Aspergillus niger. When the host cell according to thedisclosure is an Aspergillus niger host cell, the host cell preferablyis CBS 513.88, CBS124.903 or a derivative thereof.

Several strains of filamentous fungi are readily accessible to thepublic in a number of culture collections, such as the American TypeCulture Collection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS),Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL), and All-Russian Collection ofMicroorganisms of Russian Academy of Sciences, (abbreviation inRussian—VKM, abbreviation in English—RCM), Moscow, Russia. Usefulstrains in the context of the present disclosure may be Aspergillusniger CBS 513.88, CBS124.903, Aspergillus oryzae ATCC 20423, IFO 4177,ATCC 1011, CBS205.89, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892,P. chrysogenum CBS 455.95, P. chrysogenum Wisconsin54-1255(ATCC28089),Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2, Thielaviaterrestris NRRL8126, Talaromyces emersonii CBS 124.902, Acremoniumchrysogenum ATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 orATCC 56765 or ATCC 26921, Aspergillus sojae ATCC11906, Myceliophthorathermophila C1, Garg 27K, VKM-F 3500 D, Chrysosporium lucknowense C1,Garg 27K, VKM-F 3500 D, ATCC44006 and derivatives thereof.

A host cell may be a recombinant or transgenic host cell. The host cellmay be genetically modified with a nucleic acid construct or expressionvector as disclosed herein with standard techniques known in the art,such as electroporation, protoplast transformation or conjugation forinstance as disclosed in Sambrook & Russell, Molecular Cloning: ALaboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, N.Y., 2001.

Standard genetic techniques, such as overexpression of enzymes in thehost cells, genetic modification of host cells, or hybridisationtechniques, are known methods in the art, such as described in Sambrookand Russel (2001) “Molecular Cloning: A Laboratory Manual (3^(rd)edition), Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, or F. Ausubel et al, eds., “Current protocols in molecularbiology”, Green Publishing and Wiley Interscience, New York (1987).Methods for transformation, genetic modification etc of fungal hostcells are known from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 andWO 00/37671, WO90/14423, EP-A-0481008, EP-A-0635 574 and U.S. Pat. No.6,265,186.

The disclosure provides a method of producing a lipolytic polypeptidevariant, which method comprises:

-   -   a) selecting a polypeptide having lipolytic activity;    -   b) substituting at least one amino acid residue corresponding to        any of 53, 112, 141, 178, 179, 182, 202, 203, 235, 282, 284,        -   said positions being defined with reference to SEQ ID NO: 2;    -   c) optionally substituting one or more further amino acids as        defined in b);    -   d) preparing the variant resulting from steps a)-c);    -   e) determining a property of the variant; and    -   f) selecting a variant having an altered property in comparison        with a reference polypeptide having lipolytic activity measured        under the same conditions, thereby to produce a lipolytic        polypeptide variant.        The disclosure provides a method of producing a lipolytic        polypeptide variant, which method comprises:    -   a) selecting a polypeptide having lipolytic activity;    -   b) substituting at least one amino acid residue corresponding to        any of 53, 112, 141, 178, 179, 182, 202, 203, 235, 282, 284,        -   said positions being defined with reference to SEQ ID NO: 2;    -   c) optionally substituting one or more further amino acids as        defined in b);    -   d) preparing the variant resulting from steps a)-c);    -   e) determining a property of the variant; and    -   f) selecting a variant having an altered property in comparison        with a reference polypeptide having lipolytic activity, thereby        to produce a lipolytic polypeptide variant measured under the        same conditions wherein the altered property includes an altered        ratio of the activity on esters of long chain fatty acids: the        activity on esters of short chain fatty acids as compared to the        reference polypeptide having lipolytic activity measured under        the same conditions.        The disclosure provides a method of producing a lipolytic        polypeptide variant, which method comprises:    -   a) selecting a polypeptide having lipolytic activity;    -   b) substituting at least one amino acid residue corresponding to        any of 53, 112, 141, 178, 179, 182, 202, 203, 235, 282, 284,        -   said positions being defined with reference to SEQ ID NO: 2;    -   c) optionally substituting one or more further amino acids as        defined in b);    -   d) preparing the variant resulting from steps a)-c);    -   e) determining a property of the variant; and    -   f) selecting a variant having an altered property in comparison        with a reference polypeptide having lipolytic activity, thereby        to produce a lipolytic polypeptide variant, wherein the altered        property includes an increased ratio of the activity on esters        of long chain fatty acids: the activity on esters of short chain        fatty acids as compared to the reference polypeptide having        lipolytic activity measured under the same conditions.

The disclosure provides a method of producing a lipolytic polypeptidevariant, wherein in step b) the substitution, when aligned with theamino acid sequence as set out in SEQ ID NO: 2, comprises one or more of

-   -   Y53S    -   S112T    -   F141M    -   A178G    -   V179L    -   V179M    -   L182F    -   P202A    -   R203M    -   P235L    -   L282F    -   I284Q        said positions being defined with reference to SEQ ID NO: 2, and        wherein the variant has one or more altered properties as        compared with a reference polypeptide having lipolytic activity.

The disclosure provides a method of producing a lipolytic polypeptidevariant, wherein in step b) the substitution, when aligned with theamino acid sequence as set out in SEQ ID NO: 2, comprises one or more of

Y53S

S112T

F141M

A178G

V179L

V179M

L182F

P202A

R203M

P235L

L282F

I284Q

said positions being defined with reference to SEQ ID NO: 2,

and wherein the variant has an increased ratio of the activity on estersof long chain fatty acids: the activity on esters of short chain fattyacids as compared with a reference polypeptide having lipolyticactivity.

The disclosure provides a composition comprising the variant polypeptideaccording to the disclosure or obtainable by the method according to thedisclosure and one or more components selected from the group consistingof milk powder, gluten, granulated fat, an additional enzyme, an aminoacid, a salt, an oxidant, a reducing agent, an emulsifier, sodiumstearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters offatty acids and diacetyl tartaric acid esters of mono- and diglycerides,a gum, a flavour, an acid, a starch, a modified starch, a humectant anda preservative.

A composition includes a pre-mix. The term “pre-mix” is defined hereinto be understood in its conventional meaning, i.e. as a mix of bakingagents, generally including flour, which may be used not only inindustrial bread-baking plants/facilities, but also in retail bakeries.The pre-mix according to the disclosure may be prepared by mixing thelipolytic polypeptide variant according to the disclosure polypeptidewith a suitable carrier such as flour (e.g. wheat flour, corn flourand/or rice flour), starch, maltodextrin or a salt. The pre-mix maycontain additives as mentioned herein.

Additives are in most cases added in powder form. Suitable additivesinclude oxidants (including ascorbic acid, bromate and azodicarbonamide(ADA), reducing agents (including L-cysteine), emulsifiers (includingwithout limitation mono- and diglycerides, monoglycerides such asglycerol monostearate (GMS), sodium stearoyl lactylate (SSL), calciumstearoyl lactylate (CSL), polyglycerol esters of fatty acids (PGE) anddiacetyl tartaric acid esters of mono- and diglycerides (DATEM),propylene glycol monostearate (PGMS), lecithin), gums (including guargumand xanthangum), flavours, acids (including citric acid, propionicacid), starches, modified starches, humectants (including glycerol) andpreservatives.

In a composition according to the disclosure the additional enzyme isselected from a further lipolytic enzyme; an amylase such as analpha-amylase, for example a fungal alpha-amylase (which may be usefulfor providing sugars fermentable by yeast), a beta-amylase; aglucanotransferase; a peptidase in particular, an exopeptidase (whichmay be useful in flavour enhancement); a transglutaminase; a cellulase;a hemicellulase, in particular a pentosanase such as xylanase (which maybe useful for the partial hydrolysis of pentosans, more specificallyarabinoxylan, which increases the extensibility of the dough); protease(which may be useful for gluten weakening in particular when using hardwheat flour); a protein disulfide isomerase, e.g., a protein disulfideisomerase as disclosed in WO 95/00636; a glycosyltransferase; aperoxidase (which may be useful for improving the dough consistency); alaccase; an oxidase, such as an hexose oxidase, a glucose oxidase,aldose oxidase, pyranose oxidase; a lipoxygenase; L-amino acid oxidase(which may be useful in improving dough consistency) and anasparaginase.

In an embodiment of the composition according to the disclosure theadditional enzyme is a further lipolytic enzyme, i.e. an enzyme thathydrolyses triacylglycerol and/or galactolipid and/or phospholipids.

The further lipolytic enzyme may be a lipolytic enzyme as described inWO2009/106575, such as the commercial product Panamore®, product of DSM.

The further lipolytic enzyme may be a mammalian phospholipase such aspancreatic PLA2, e.g. bovine or porcine PLA2 such as the commercialproduct Lecitase 10L (porcine PLA2, product of Novozymes A/S).

The further lipolytic enzyme may be from Fusarium, e.g. F. oxysporumphospholipase A1 (WO 1998/026057), F. venenatum phospholipase A1(described in WO 2004/097012 as a phospholipase A2 called FvPLA2), fromTuber, e.g. T. borchii phospholipase A2 (called TbPLA2, WO 2004/097012).The further lipolytic enzyme may be as described in WO 2000/032758 or WO2003/060112.

Panamore®, Lipopan® F, Lipopan® 50 and Lipopan® S are commercialised tostandardised lipolytic activity, using a measurement of DLU forPanamore® from DSM and a measurement of LU for the Lipopan® family fromNovozymes. DLU is defined as the amount of enzyme needed to produce 1micromol/min of p-nitrophenol from p-nitrophenyl palmitate at pH 8.5 at37° C., while LU is defined as the amount of enzyme needed to produce 1micromol/min of butyric acid from tributyrin at pH 7 at 30° C. Thefurther lipolytic enzyme may be used at 2-850 DLU/kg flour or at50-23500 LU/kg flour. The further enzyme may be used at 14 DLU to 164DLU/kg flour, in an aspect 27-77 DLU/kg flour.

In an embodiment of the composition according to the disclosure thefurther lipolytic enzyme is a lipolytic enzyme having at least 60%identity to the amino acid sequence as set out in amino acids 34 to 304of SEQ ID NO 2, in an aspect the further lipolytic enzyme is a lipolyticenzyme comprising an amino acids sequence as set out in amino acids 34to 304 of SEQ ID NO 2.

In an embodiment of the enzyme composition of the disclosure theadditional enzyme is an enzyme as claimed in EP0869167B1.

In an embodiment of the composition according to the disclosure theadditional enzyme is an alpha-amylase having at least 70% identity tothe amino acid sequence as set out in SEQ ID NO: 4, in an aspect theadditional enzyme is an alpha-amylase comprising an amino acid sequenceas set out in SEQ ID NO: 4.

In an embodiment of the composition according to the disclosurecomprises a lipolytic enzyme variant according to the disclosure and analpha-amylase having at least 70% identity to the amino acid sequence asset out in SEQ ID NO 4.

In an embodiment of the composition according to the disclosure theadditional enzyme is an amylase having at least 70% identity to theamino acid sequence as set out in SEQ ID NO 5, in an aspect theadditional enzyme is an amylase comprising an amino acid sequence as setout in SEQ ID NO: 5.

In an embodiment of the composition according to the disclosurecomprises a lipolytic enzyme variant according to the disclosure and anamylase having at least 70% identity to the amino acid sequence as setout in SEQ ID NO: 5.

In an aspect of the enzyme composition according to the disclosure theadditional enzyme is an enzyme as described in WO 9943794, in particularas claimed in EP1058724B1.

In an embodiment of the composition according to the disclosure theadditional enzyme is an amylase having an amino acid sequence at least70% identical to the amino acid sequence as set out in SEQ ID NO: 6, inan aspect the additional enzyme is an amylase comprising an amino acidsequence as set out in SEQ ID NO: 6.

In an embodiment of the composition according to the disclosurecomprises a lipolytic enzyme variant according to the disclosure and anamylase having at least 70% identity to the amino acid sequence as setout in SEQ ID NO: 6.

In an embodiment of the composition according to the disclosure theadditional enzyme is an amylase having an amino acid sequence at least70% identical to the amino acid sequence as set out in SEQ ID NO: 7, inan aspect the additional enzyme is an amylase comprising an amino acidsequence as set out in SEQ ID NO: 7.

In an embodiment of the composition according to the disclosurecomprises a lipolytic enzyme variant according to the disclosure and anamylase having at least 70% identity to the amino acid sequence as setout in SEQ ID NO: 7.

The disclosure provides a composition comprising the variant polypeptideaccording to the disclosure or obtainable by the method according to thedisclosure and a hemicellulase, such as a xylanase.

The disclosure provides a composition comprising the variant polypeptideaccording to the disclosure or obtainable by the method according to thedisclosure and DATEM.

The disclosure provides a use of the variant polypeptide according tothe disclosure, or of the composition according to the disclosure, inthe production of a food product, preferably in the production of adough and/or a baked product.

Use of the Lipolytic Enzyme in Industrial Processes

The disclosure also relates to the use of the lipolytic enzyme variantaccording to the disclosure in a number of industrial processes. Despitethe long-term experience obtained with these processes, the lipolyticenzyme variant according to the disclosure features a number ofsignificant advantages over the enzymes currently used. Depending on thespecific application, these advantages can include aspects like lowerproduction costs, higher specificity towards the substrate, lessantigenic, less undesirable side activities, higher yields when producedin a suitable microorganism, more suitable pH and temperature ranges,better tastes of the final product as well as food grade and kosheraspects.

Preferably the lipolytic enzyme variant according to the disclosure canbe used in the food industry, more preferably in food manufacturing.

Use in Industrial Applications Bakery Applications

An example of an industrial application of the lipolytic enzyme variantaccording to the disclosure in food is its use in baking applications toimprove properties of a dough and or a baked product. It has been foundthat the lipolytic enzyme variants according to the disclosure can actupon several types of lipids, ranging from glycerides (e.g.triglycerides), phospholipids, and/or glycolipids, such asgalactolipids, which are relevant in bakery applications. This isadvantageous when used as a replacer of chemical emulsifiers in dough.

The use of the lipolytic enzyme variant according to the disclosure inbaking applications modifies the natural flour lipids. This may resultin improved stabilization of the dough. It may ensure a more stabledough in case of over-proofing, a larger loaf volume, and/or improvedcrumb structure. An improved crumb structure includes that the crumbstructure may become more uniform and with smaller crumb cells, thecrumb texture may become silkier and/or the crumb colour may appear tobe whiter. The use of the lipolytic enzyme variant according to thedisclosure in the baking industry preferably reduces the need foraddition of emulsifiers like DATEM, CSL, PGME, PGE and/or SSL thatotherwise are commonly added to dough for example to stabilise it.

Dough

The term “dough” is defined herein as a mixture of flour and otheringredients. In one aspect the dough is firm enough to knead or roll.The dough may be fresh, frozen, prepared or parbaked. The preparation offrozen dough is described by Kulp and Lorenz in Frozen and RefrigeratedDoughs and Batters.

Dough is made using dough ingredients, which include without limitation(cereal) flour, a lecithin source including egg, water, salt, sugar,flavours, a fat source including butter, margarine, oil and shortening,baker's yeast, chemical leavening systems such as a combination of anacid (generating compound) and bicarbonate, a protein source includingmilk, soy flour, oxidants (including ascorbic acid, bromate andazodicarbonamide (ADA)), reducing agents (including L-cysteine),emulsifiers (including mono/di glycerides, monoglycerides such asglycerol monostearate (GMS), sodium stearoyl lactylate (SSL), calciumstearoyl lactylate (CSL), polyglycerol esters of fatty acids (PGE) anddiacetyl tartaric acid esters of mono- and diglycerides (DATEM), gums(including guargum and xanthangum), flavours, acids (including citricacid, propionic acid), starch, modified starch, gluten, humectants(including glycerol) and preservatives.

For leavened products, primarily baker's yeast is used next to chemicalleavening systems such as a combination of an acid (generating compound)and bicarbonate.

Cereals include maize, rice, wheat, barley, sorghum, millet, oats, rye,triticale, buckwheat, quinoa, spelt, einkorn, emmer, durum and kamut.

Dough is usually made from basic dough ingredients including (cereal)flour, such as wheat flour or rice flour, water and optionally salt. Forleavened products, primarily baker's yeast is used next to chemicalleavening systems such as a combination of an acid (generating compound)and bicarbonate.

The term dough herein includes a batter. A batter is a semi-liquidmixture, being thin enough to drop or pour from a spoon, of one or moreflours combined with liquids such as water, milk or eggs used to preparevarious foods, including cake.

The dough may be made using a mix including a cake mix, a biscuit mix, abrownie mix, a bread mix, a pancake mix and a crepe mix.

The term dough includes retarded dough, here the dough is stored at orbelow 5° C. during a storage period and recovered for bake off andsales.

The term retarded dough, such as frozen dough. The frozen dough istypically used for manufacturing baked products including withoutlimitation biscuits, breads, bread sticks and croissants.

Frozen dough has several advantages for the bakery industry such as areduction of night labour, better flexibility in production and allowingbakeries to offer a broader assortment of fresh breads.

In an aspect the disclosure relates to the use of the lipolytic enzymevariant of the disclosure in the preparation of a frozen dough.

A further aspect includes the use of the lipolytic enzyme variant of thedisclosure for improving the crumb structure of a baked product preparedfrom the frozen dough.

A further aspect includes the use of the lipolytic enzyme variant of thedisclosure for increasing the dough stability of the frozen dough.

In an aspect the disclosure relates to the use of the lipolytic enzymevariant of the disclosure in the preparation of a baked product producedusing whole-meal flour.

A further aspect includes the use of the lipolytic enzyme variant of thedisclosure for improving the crumb structure of a baked product producedusing whole-meal flour.

A further aspect includes the use of the lipolytic enzyme variant of thedisclosure for increasing the dough stability of a baked productproduced using whole-meal flour.

A further aspect includes the use of the lipolytic enzyme variant of thedisclosure for increasing the dough stability of a baked productproduced using whole-meal flour.

Baked Product

The term ‘baked product’ refers to a baked food product prepared from adough.

Examples of baked products, whether of a white, brown or whole-mealtype, which may be advantageously produced by the present disclosureinclude bread (in particular white, whole-meal or rye bread), typicallyin the form of loaves or rolls, French baguette-type bread, pastries,croissants, brioche, panettone, pasta, noodles (boiled or (stir-)fried),pita bread and other flat breads, tortillas, tacos, cakes, pancakes,cookies in particular biscuits, doughnuts, including yeasted doughnuts,bagels, pie crusts, steamed bread, crisp bread, brownies, sheet cakes,snack foods (e.g., pretzels, tortilla chips, fabricated snacks,fabricated potato crisps). The term baked product includes withoutlimitation, bread containing from 2 to 30 wt % sugar based on totalrecipe weight, fruit containing bread, breakfast cereals, cereal bars,eggless cake, soft rolls and gluten-free bread.

The term baked product includes without limitation, bread containingfrom 2 to 30 wt % sugar based on total recipe weight, fruit containingbread, breakfast cereals, cereal bars, eggless cake, soft rolls,gluten-free bread, cake, doughnuts, brioche, hamburger buns, Brusselswaffles. In an aspect the baked products is a baked product comprisingsucrose such as bread containing from 2 to 30 wt % sugar based on totalrecipe weight, cake, doughnuts, brioche, hamburger buns, Brusselswaffles. Gluten-free bread herein and herein after is bread thatcontains at most 20 ppm gluten. Several grains and starch sources areconsidered acceptable for a gluten-free diet. Frequently used sourcesare potatoes, rice and tapioca (derived from cassava). A baked productherein includes without limitation tin bread, loaves of bread, twists,buns, such as hamburger buns or steamed buns, chapati, rusk, dried steambun slice, bread crumb, matzos, focaccia, melba toast, zwieback,croutons, soft pretzels, soft and hard bread, bread sticks, yeastleavened and chemically-leavened bread, laminated dough products such asDanish pastry, croissants or puff pastry products, muffins, bagels,confectionery coatings, crackers, wafers, pizza crusts, tortillas, pastaproducts, crepes, waffles, parbaked products. An example of a parbakedproduct includes, without limitation, partially baked bread that iscompleted at point of sale or consumption with a short second bakingprocess. A baked product herein includes without limitation pound cake,butter cake, sponge cake, muffin, biscuit cake, roulade, genoise andchiffon cake, foam cakes.

The bread may be white or brown pan bread; such bread may for example bemanufactured using a so called American style Sponge and Dough method oran American style Direct method. The bread may be a sour dough bread.

The term tortilla herein includes corn tortilla and wheat tortilla. Acorn tortilla is a type of thin, flat bread, usually unleavened madefrom finely ground maize (usually called “corn” in the United States). Aflour tortilla is a type of thin, flat bread, usually unleavened, madefrom finely ground wheat flour. The term tortilla further includes asimilar bread from South America called arepa, though arepas aretypically much thicker than tortillas. The term tortilla furtherincludes a laobing, a pizza-shaped thick “pancake” from China and anIndian Roti, which is made essentially from wheat flour. A tortillausually has a round or oval shape and may vary in diameter from about 6to 30 cm.

The disclosure provides a use wherein the use comprises replacing atleast part of a chemical emulsifier in the production of a dough and/ora baked product.

Such use may be to fully replace a chemical emulsifier in themanufacturing of a dough and/or a baked product. The use may be toreplace at least part of DATEM in the manufacturing of a dough and/or abaked product. The lipolytic enzyme variant according to the disclosuremay be used to fully or partially replace the emulsifier DATEM. DATEM isthe acronym for diacetyl tartaric acid esters of mono- and diglycerides.One of the main components in DATEM may be1-stearoyl-3-diacetyltartryl-glycerol.

The use may be to replace at least part of SSL and/or CSL in themanufacturing of a dough and/or baked product.

The disclosure provides a use wherein the use is to the manufacturing alow chemical emulsifier or chemical emulsifier free baked product.

A low chemical emulsifier baked product is a baked product, such asbread comprising below 0.3 weight % based on flour chemical emulsifier(such as DATEM, SSL and/or CSL).

Emulsifier free indicates zero use of chemical emulsifier.

The disclosure provides a dough comprising the variant polypeptideaccording to the disclosure, a variant polypeptide obtainable by themethod according to the disclosure or the composition according to thedisclosure.

The preparation of a dough from the dough ingredients is well known inthe art and includes mixing of said dough ingredients and optionally oneor more moulding and leavening steps.

Preparing a dough according to the disclosure may comprise the step ofcombining the lipolytic enzyme variant according to the disclosure orthe composition according to the disclosure or the pre-mix according tothe disclosure and at least one dough ingredient. ‘Combining’ includeswithout limitation, adding the polypeptide or the composition accordingto the disclosure to the at least one dough ingredient, adding the atleast one dough ingredient to the lipolytic enzyme variant or thecomposition according to the disclosure, mixing the lipolytic enzymevariant according to the disclosure and the at least one doughingredient.

Enzymes may be combined with the at least one dough ingredient in a dry,e.g. granulated form, in a liquid form, in tablet form or in the form ofa paste. Additives are in most cases added in powder form. A granulateform or agglomerated powder comprising the lipolytic enzyme variantaccording to the disclosure preferably has a narrow particle sizedistribution with more than 95% (by weight) of the particles in therange from 25 to 500 μm.

The lipolytic enzyme variant according the disclosure may be added to adough, any ingredient from which the dough is to be made, and/or anymixture of dough ingredients from which the dough is to be made. Inother words, the lipolytic enzyme variant according to the disclosuremay be added in any step of the dough preparation and may be added inone, two or more steps.

The incorporation of an effective amount of the lipolytic enzymeaccording to the disclosure in a dough preferably reduces the need foraddition of emulsifiers like DATEM and/or SSL that otherwise arecommonly added to dough in order to stabilise it.

The term “effective amount” is defined herein as an amount of thelipolytic enzyme variant according to the disclosure that is sufficientfor providing a measurable effect on at least one property of interestof the dough and/or baked product.

An effective amount of the composition according to the disclosure adefined herein as an amount of the composition according to thedisclosure that is sufficient for providing a measurable effect on atleast one property of interest of the dough and/or baked product. Aneffective amount for bread of the lipolytic enzyme variant includes10-50 DLU per kg flour. An effective amount for bread of the lipolyticenzyme variant includes 400-700 DLU per kg batter (total weight of theingredients).

DLU is defined as the amount of enzyme needed to produce 1 micromol/minof p-nitrophenol from p-nitrophenyl palmitate at pH 8.5 at 37° C. DLUmay be measured analogously to as described in Assay 8A.

The disclosure provides a process for the production a dough comprisingthe step of combining an effective amount of the variant polypeptideaccording to the disclosure, an effective amount of the variantpolypeptide obtainable by the method according to the disclosure oreffective amount of the composition according to the disclosure with atleast one dough ingredient.

If one or more additional enzymes are used in the process for theproduction a dough, these may be added separately or together with thelipolytic enzyme variant according to the disclosure, optionally asconstituent(s) of the bread-improving and/or dough-improvingcomposition. The additional enzymes may be dosed in accordance withestablished baking practices.

The disclosure provides a process for the production of a baked product,which method comprises baking the dough according to the disclosure orthe dough obtained by the process of the disclosure.

The lipolytic enzyme variant and/or the composition according to thedisclosure may be used in the preparation of a wide range of cakes,including shortened cakes, such as for example pound cake and buttercake, and including foam cakes, such as for example meringues, spongecake, biscuit cake, roulade, genoise and chiffon cake. The lipolyticenzyme variant and/or the composition according to the disclosure may beused in the preparation of a muffin. Sponge cake is a highly aeratedtype of soft cake based on wheat flour, sugar and eggs (and optionallybaking powder and fat or oil). It is often used as a base for othertypes of cakes and desserts. A pound cake is traditionally prepared fromone pound each of flour, butter, eggs, and sugar, optionallycomplemented with baking powder. Sugar and egg yolk is decreasedcompared to pound or sponge cake and egg white content is increased.

A method to prepare a batter preferably comprises the steps of:

a. preparing the batter of the cake by adding at least:

-   -   i. sugar;    -   ii. flour;    -   iii. the lipolytic enzyme variant according to the disclosure;    -   iv. at least one egg; and    -   v. optionally a fat.        Fat includes, butter margarine, oil, and shortening. Optional        ingredients include starch, milk components and/or emulsifier.        A method to prepare a cake according to the disclosure further        comprises the step of

b. baking the batter to yield a cake.

The person skilled in the art knows how to prepare a batter or a cakestarting from dough ingredients.

The lipolytic enzyme variant according to the disclosure may be usedboth in regular cakes and in cakes in which the amount of eggs and/orfat has been reduced. The reduction of the amount of eggs and/or fatwhich is possible differs per type of cake. The person skilled in theart knows the amount of eggs and/or fat which are regularly present incake recipes and which is dependent on the type of cake. For example areduction of the amount of eggs of at least 5% w/w based on total weightof the batter may be reached. In an aspect a reduction of the amount ofeggs of at least 10% w/w may be reached, in a further aspect a reductionof at least 20% w/w of the amount of eggs may be reached. In general areduction of the amount of fat of at least 10% w/w based on total weightof the batter may be reached. In an aspect a reduction of the amount offat of at least 20% may be reached. In a further aspect a reduction ofat least 30% of fat may be reached.

The lipolytic enzyme variant according to the disclosure may be used toreduce the amount of egg in the preparation of cake.

In an aspect of the method to prepare a batter, the batter comprisesbetween 3 and 25 wt % whole egg based on the total weight of the batter.In an aspect of the method to prepare a batter, the batter comprisesbetween 4 and 20 wt % whole egg based on the total weight of the batter.In an aspect of the method to prepare a batter, the batter comprisesbetween 5 and 15 wt % whole egg based on the total weight of the batter.In an aspect of the method to prepare a batter, the batter comprisesbetween 6 and 12 wt % whole egg based on the total weight of the batter.

Embodiments of the Disclosure

1. A variant polypeptide having lipolytic activity, wherein the varianthas an amino acid sequence which, when aligned with the amino acidsequence as set out in SEQ ID NO: 2, comprises at least one substitutionof an amino acid residue at a position corresponding to any of thepositions 53, 112, 141, 178, 179, 182, 202, 203, 235, 282, 284,

said positions being defined with reference to SEQ ID NO: 2,

and wherein said variant has at least 70% identity with the maturepolypeptide having lipolytic activity as set out in SEQ ID NO: 2.

2. A variant polypeptide according to embodiment 1, wherein thereference polypeptide comprises the mature lipolytic enzyme as set outin SEQ ID NO: 2.

3. A variant polypeptide according to embodiment 1 or 2, wherein thereference polypeptide is the mature lipolytic enzyme as set out in SEQID NO: 2.

4. A variant polypeptide according to any one of embodiments 1 to 3,wherein the mature polypeptide comprises an amino acid sequence as setout in amino acids 34 to 304 of SEQ ID NO: 2.

5. A variant polypeptide according to any one of embodiments 1 to 4,wherein the mature polypeptide has an amino acid sequence as set out inamino acids 34 to 304 of SEQ ID NO: 2.

6. A variant polypeptide according to any one of embodiments 1 to 5,wherein the variant has one or more altered properties as compared witha reference polypeptide having lipolytic activity.

7. A variant polypeptide according to any one of embodiments 1 to 6which, when aligned with the amino acid sequence as set out in SEQ IDNO: 2, comprises at least one substitution of an amino acid residue at aposition corresponding to any of the positions

179, 282, 284.

8. A variant polypeptide according to any one of embodiments 1 to 7,wherein the variant demonstrates an altered ratio of the activity onesters of long chain fatty acids: the activity on esters of short chainfatty acids as compared to the reference polypeptide measured under thesame conditions.

9. A variant polypeptide according to any one of embodiments 1 to 8,wherein the variant demonstrates an increased ratio of the activity onesters of long chain fatty acids: the activity on esters of short chainfatty acids as compared to the reference polypeptide measured under thesame conditions.

10. A variant polypeptide according to any one of embodiments 1 to 9which, when aligned with the amino acid sequence as set out in SEQ IDNO: 2, comprises at least one substitution of an amino acid residue at aposition corresponding to any of the positions 53, 203.

11. A variant polypeptide according to any one of embodiments 1 to 9which, when aligned with the amino acid sequence as set out in SEQ IDNO: 2, comprises at least one substitution of an amino acid residue at aposition corresponding to any of the positions 179, 282, 284.

12. A variant polypeptide according to any one of embodiments 1 to 11which, when aligned with the amino acid sequence as set out in SEQ IDNO: 2, comprises at least one substitution of an amino acid residue at aposition corresponding to any of the positions 53, 112, 141, 178, 179,182, 203, 235, and wherein the variant demonstrates an increased ratioof the activity on esters of long chain fatty acids: the activity onesters of short chain fatty acids determined at pH 5.5 as compared tothe reference polypeptide having lipolytic activity.

13. A variant polypeptide according to any one of embodiments 1 to 12which, when aligned with the amino acid sequence as set out in SEQ IDNO: 2, comprises at least one substitution of an amino acid residue at aposition corresponding to any of the positions 53, 112, 141, 178, 179,202, 203, 235, 282, 284, and wherein the variant demonstrates anincreased ratio of the activity on esters of long chain fatty acids: theactivity on esters of short chain fatty acids determined at pH 7 ascompared to the reference polypeptide having lipolytic activity.

14. A variant polypeptide according to any one of embodiments 1 to 13which, when aligned with the amino acid sequence as set out in SEQ IDNO: 2, comprises one or more of the amino acid substitutions selectedfrom

Y53S

S112T

F141M

A178G

V179L

V179M

L182F

P202A

R203M

P235L

L282F

I284Q

15. A variant polypeptide according to any one of embodiments 1 to 14which, when aligned with the amino acid sequence as set out in SEQ IDNO: 2, comprises one or more of the amino acid substitutions selectedfrom

V179L

V179M

L282F

I284Q

16. A nucleic acid sequence encoding a variant polypeptide according toany one of the preceding embodiments.

17. A nucleic acid construct comprising the nucleic acid sequence ofembodiment 16 operably linked to one or more control sequences capableof directing the expression of a lipolytic enzyme in a suitableexpression host.

18. A recombinant expression vector comprising the nucleic acidconstruct of embodiment 17.

19. A recombinant host cell comprising the expression vector ofembodiment 18.

20. A method for producing a lipolytic polypeptide variant according toany one of embodiments 1 to 15 comprising cultivating the host cell ofembodiment 19 under conditions conducive to production of the lipolyticenzyme variant and recovering the lipolytic enzyme variant.

21. A method of producing a lipolytic polypeptide variant, which methodcomprises:

a) selecting a polypeptide having lipolytic activity;

b) substituting at least one amino acid residue corresponding to any of

-   -   53, 112, 141, 178, 179, 182, 202, 203, 235, 282, 284,    -   said positions being defined with reference to SEQ ID NO: 2;

c) optionally substituting one or more further amino acids as defined inb);

d) preparing the variant resulting from steps a)-c);

e) determining a property of the variant; and

f) selecting a variant having an altered property in comparison with areference polypeptide having lipolytic activity measured under the sameconditions, thereby to produce a lipolytic polypeptide variant.

22. The method according to embodiment 21, wherein in steps e) and f)comprise:

e) determining the ratio of the activity on esters of long chain fattyacids: the activity on esters of short chain fatty acids of the variantand of a reference polypeptide having lipolytic activity; and

f) selecting a variant having an altered ratio of the activity on estersof long chain fatty acids: the activity on esters of short chain fattyacids in comparison to the reference polypeptide, thereby to produce alipolytic polypeptide variant.

23. The method according to embodiment 21 or 22, wherein step f)comprises:

f) selecting a variant having an increased ratio of the activity onesters of long chain fatty acids: the activity on esters of short chainfatty acids in comparison to the reference polypeptide, thereby toproduce a lipolytic polypeptide variant.

24. The method according to any one of embodiments 21 to 23 wherein instep b) the substitution, when aligned with the amino acid sequence asset out in SEQ ID NO: 2, comprises one or more of

Y53S

S112T

F141M

A178G

V179L

V179M

L182F

P202A

R203M

P235L

L282F

I284Q

said positions being defined with reference to SEQ ID NO: 2,

and wherein the variant has one or more altered properties as comparedwith a reference polypeptide having lipolytic activity.

25. A composition comprising the variant polypeptide according to anyone of embodiments

1 to 15 or obtainable by the method according to any one of embodiments20 to 24 and one or more components selected from the group consistingof milk powder, gluten, granulated fat, an additional enzyme, an aminoacid, a salt, an oxidant, a reducing agent, an emulsifier, sodiumstearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters offatty acids and diacetyl tartaric acid esters of mono- and diglycerides,a gum, a flavour, an acid, a starch, a modified starch, a humectant anda preservative.

26. A composition according embodiment 25 wherein the additional enzymeis selected from a further lipolytic enzyme; an amylase such as analpha-amylase, for example a fungal alpha-amylase, a beta-amylase; aglucanotransferase; a peptidase in particular, an exopeptidase; atransglutaminase; a cellulase; a hemicellulase, in particular apentosanase such as xylanase; protease; a protein disulfide isomerase; aglycosyltransferase; a peroxidase; a laccase; an oxidase, such as anhexose oxidase, a glucose oxidase, aldose oxidase, pyranose oxidase; alipoxygenase; L-amino acid oxidase and an asparaginase.

27. A pre-mix comprising flour and the variant polypeptide according toany one of embodiments 1 to 15, the variant polypeptide obtainable bythe method according to anyone of embodiments 20 to 24 or thecomposition according to embodiment 25 or 26.

28. Use of the variant polypeptide according to any one of embodiments 1to 15, or of the composition according to any one of embodiment 25 or26, or of the pre-mix according to embodiment 27 in the manufacturing ofa food product, preferably in the manufacturing of a dough and/or abaked product.

29. Use according to embodiment 28, wherein the use comprises replacingat least part of a chemical emulsifier in the manufacturing of a doughand/or a baked product.

30. Use according to embodiment 28, wherein the use is to fully replacea chemical emulsifier in the manufacturing of a dough and/or a bakedproduct.

31. The use according to embodiment 29 or 30, wherein the use comprisesreplacing at least part of DATEM in the manufacturing of a dough and/ora baked product.

32. The use according to embodiment 29 or 30, wherein the use comprisesreplacing at least part of SSL and/or CSL in the manufacturing of adough and/or baked product.

33. Use of the variant polypeptide according to any one of embodiments 1to 15, or of the composition according to any one of embodiment 25 or26, or of the pre-mix according to embodiment 24 in the manufacturing ofa low chemical emulsifier or chemical emulsifier free baked product.

34. The use according to embodiment 33, wherein the baked product hasbeen produced using whole-meal flour and/or whole grain.

35. The use according to any one of embodiments 28 to 34 wherein thebaked product has been produced from a frozen dough.

36. The use according to embodiment 33 and/or 34 wherein the chemicalemulsifier is mono- and diglycerides of fatty acids or distilled monoglycerides sodium stearoyl lactylate (SSL), calcium stearoyl lactylate(CSL), polyglycerol esters of fatty acids (PGE), propylene glycol monoesters of fatty acids (PGME), and diacetyl tartaric acid esters of mono-and diglycerides (DATEM) or lecithin.

37. The use according to any one of embodiments 28 to 36, wherein theuse is to increase volume of a baked product.

38. The use according to any one of embodiments 28 to 36, wherein theuse is to reduce hardness of a baked product.

39. The use according to any one of embodiments 28 to 36, wherein theuse is to create a finer crumb structure of a baked product.

40. The use according to any one of embodiments 28 to 36, wherein theuse is to increase stability of a dough.

41. The use according to any one of embodiments 28 to 36, wherein thebaked product is bread, cake and/or pastry.

42. The use according to any one of embodiments 28 to 36, 41 wherein theuse is to reduce the amount of egg in the preparation of cake.

43. A dough comprising a variant polypeptide according to any one ofembodiments 1 to 15, the variant polypeptide obtainable by the methodaccording to any one of embodiments 20 to 24, the composition accordingto embodiment 25 or 26, or the pre-mix according to embodiment 27.

44. A process for preparing a dough comprising the step of combining aneffective amount of the variant polypeptide according to any one ofembodiments 1 to 15, an effective amount of the variant polypeptideobtainable by the method according to any one of embodiments 20 to 24,an effective amount of the composition according to embodiment 25 or 26or an effective amount of the pre-mix according to embodiment 27 with atleast one dough ingredient.

45. A process for the production of a baked product, which methodcomprises baking the dough according to embodiment 43 or the doughobtained by the process of embodiment 44.

46. The process according to embodiment 45, wherein the baked product isbread, cake and/or pastry.

47. A baked product obtainable by the process according to embodiment 45or 46, or by the use according to any one of embodiments 28 to 42.

48. A process for the production of a food product, which methodcomprises adding the variant polypeptide according to any one ofembodiments 1 to 15, the variant polypeptide obtainable by the methodaccording to any one of embodiments 20 to 24, the composition accordingto embodiment 25 or 26 or the pre-mix according to embodiment 27 to aningredient of a food product.

49. A process for the production of a food product, which methodcomprises adding an effective amount of the variant polypeptideaccording to any one of embodiments 1 to 15, an effective amount of thevariant polypeptide obtainable by the method according to any one ofembodiments 20 to 24, an effective amount of the composition accordingto embodiment 25 or 26 or an effective amount of the pre-mix accordingto embodiment 27 to an ingredient of a food product.

A reference herein to a patent document or other matter which is givenas prior art is not to be taken as an admission that that document ormatter was known or that the information it contains was part of thecommon general knowledge as at the priority date of any of the claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

The present disclosure is further illustrated by the following Examples:

EXAMPLES

General Procedures and Molecular Biology Techniques

Standard molecular cloning techniques such as DNA isolation, gelelectrophoresis, enzymatic restriction modifications of nucleic acids,E. coli transformation etc., were performed as described by Sambrook etal., 1989 (2^(nd) ed) and 2001 (3^(rd) ed), Molecular cloning: alaboratory manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. and Innes et al. (1990) PCR protocols, a guide to methodsand applications, Academic Press, San Diego. Examples of the generaldesign of expression vectors for gene overexpression and disruptionvectors for down-regulation, transformation, use of markers, strains andselective media can be found in WO199846772, WO199932617, WO2001121779,WO2005095624, WO2006040312, EP 635574B, WO2005100573, WO2011009700,WO2012001169, WO2013135729, WO2014013073 and WO2014013074. Aftertransformation, the direct repeats allow for the removal of theselection marker by a (second) homologous recombination event. Theremoval of a marker such as amdS can be done by plating onfluoro-acetamide media, resulting in the selection of marker-gene-freestrains (see also “MARKER-GENE FREE” approach in EP 0 635 574).Alternatively, a marker can be removed for example using a recombinasesuch as detailed in WO2013135729. Using these strategies oftransformation and subsequent marker removal, a marker can be usedindefinitely in strain modification programs.

Strains

WT 1: This Aspergillus niger strain is used as a wild-type strain. Thisstrain is deposited at the CBS Institute under the deposit number CBS513.88.

GBA 306: The construction of GBA 306 using WT1 as starting strain hasbeen described in detail in WO2011/009700. This GBA 306 strain has thefollowing genotype: ΔglaA, ΔpepA, ΔhdfA, an adapted BamHI amplicon,ΔamyBII, ΔamyBI, and ΔamyA.

Biochemical Assays

Materials and Methods

The reference polypeptide used in the examples is a referencepolypeptide having lipolytic activity and having an amino acid sequenceas set out in amino acids 34 to 304 of SEQ ID NO: 2.

The properties of the lipolytic enzyme variants were tested usingseveral substrates. These include the following:

-   -   Phosphatidylcholines, which may be abbreviated as PC;    -   Digalactosyldiglycerides, which may be abbreviated as DGDG;    -   Monogalactosyldiglycerides, which may be abbreviated as MGDG;        and    -   Triacylglycerols which may be abbreviated as TAG. Triolein is        applied in assays described herein as a representative of        triglycerides.    -   para-nitrophenyl linoleate (pNP-linoleate), which may also be        referred herein as an ester of C18:2 fatty acid;    -   para-nitrophenyl oleate (pNP-oleate), which may also be referred        herein as an ester of C18:1 fatty acid;    -   para-nitrophenyl stearate (pNP-stearate), which may also be        referred herein as an ester of C18:0 fatty acid;    -   para-nitrophenyl palmitate (pNP-palmitate), which may also be        referred herein as an ester of C16:0 fatty acid;    -   para-nitrophenyl butyrate (pNP-butyrate), which may also be        referred herein as an ester of C4 fatty acids;

Assay 1A Phospholipase Activity at pH 5.5

Enzymatic activity of the lipolytic enzyme variant and of the parentpolypeptide (also referred as reference polypeptide) may be expressed inNEFA Units. One Unit (U) is defined as the amount of enzyme thatliberates one micromole of free fatty acid per minute under the definedassay conditions.

The principle of the assay is as follows: A mix of enzyme, buffer,substrate, and calciumchloride is incubated at 37° C. for 10 minutes.The reaction is stopped by addition of an acidic solution. The amount offormed free fatty acids is subsequently determined using the principleof the NEFA kit (NEFA-HR (2) R1 Set, 434-91795, NEFA-HR (2) R2 Set,436-91995, NEFA standard, 270-77000, all from Wako Chemicals). Theprinciple of the NEFA method is as follows: Non-esterified fatty acid(NEFA) in the reaction sample is converted to acyl-CoA, AMP andpyrophosphoric acid (PPi) by the action of acyl-CoA synthetase (ACS), inthe presence of coenzyme A (CoA) and adenosine 5-triphosphate disodiumsalt (ATP). Acyl-CoA is oxidized to form 2,3-trans-Enoyl-CoA andhydrogen peroxide by the action of acyl-CoA oxidase (ACOD). In thepresence of peroxidase (POD), the hydrogen peroxide formed yields a bluepurple pigment after quantitative oxidation condensation with3-methyl-N-ethyl-N-(β-hydroxyethyl)-aniline (MEHA) and 4-aminoantipyrine(4-AA). The concentration of non-esterified fatty acids (NEFA)concentration is obtained by measuring absorbance of the blue purplecolor. NEFA standard consists of a solution of oleic acid, which is usedto produce a calibration line to calculate the amount of free fattyacids. The NEFA method is performed on the Konelab Arena 30 analyzer(Thermo Scientific, Vantaa, Finland) and the intensity of the color ismeasured at 540 nm. The reaction sample absorbance is corrected bysubtracting the absorbance of an appropriate reaction sample blank asdescribed below.

Enzyme samples were diluted to a range between 0.05-1.5 U/mL in 0.2 Macetate buffer pH5.5. For each reaction sample and reaction sample blankglass tubes containing 50 microliter of a 0.1 M calciumchloridesolution, 500 microliter substrate solution (1% (w/v)L-alpha-phosphatidylcholine (P3556, Sigma Aldrich) in 2% Triton X-100),and 250 microliter 0.2 M acetate buffer pH5.5 were prepared. These werepreheated for 10 minutes in a water bath at 37° C. The reaction wasstarted by adding 100 microliter of enzyme sample to the glass tube. 10minutes after enzyme sample addition, 100 microliter of 2.0 M HCl wasadded, the tube was immediately removed from the water bath and mixedwell by vortexing to terminate the reaction. For each reaction sample, acorresponding reaction sample blank was prepared by incubation ofbuffer, substrate, and calciumchloride at 37° C. After 10 minutes, 100microliter of 2.0 M HCl was added, the tube was immediately removed fromthe water bath and mixed well by vortexing. Then 100 microliter ofenzyme sample was added to the tube, and mixed well by vortexing.

Released free fatty acids were determined according to the HR SeriesNEFA-HR (2) kit instructions, which were made suitable for analyzerapplication as follows. 150 microliter of reagent NEFA-R1 was preheatedfor 300 seconds. Then 10 microliter of reaction sample was added, andincubation continued for 180 seconds. Subsequently 75 microliter ofreagent NEFA-R2 was added and incubation continued for 270 seconds. Atthis moment absorbance was measured at 540 nm. The absorbance of eachreaction sample was corrected by subtracting the absorbance of thecorresponding reaction sample blank. The amount of non-esterified fattyacids was calculated using an oleic acid calibration line.

Assay 1B Phospholipase Activity at pH 8.0

Enzymatic activity of the lipolytic enzyme variant and of the parentpolypeptide (also referred as reference polypeptide) may be expressed inNEFA Units. One Unit (U) is defined as the amount of enzyme thatliberates one micromole of free fatty acid per minute under the definedassay conditions.

The principle of the assay is as follows: A mix of enzyme, buffer,substrate, and calciumchloride is incubated at 37° C. for 10 minutes.The reaction is stopped by addition of an acidic solution. The amount offormed free fatty acids is subsequently determined using the principleof the NEFA kit (NEFA-HR (2) R1 Set, 434-91795, NEFA-HR (2) R2 Set,436-91995, NEFA standard, 270-77000, all from Wako Chemicals). Theprinciple of the NEFA method is as follows: Non-esterified fatty acid(NEFA) in the reaction sample is converted to acyl-CoA, AMP andpyrophosphoric acid (PPi) by the action of acyl-CoA synthetase (ACS), inthe presence of coenzyme A (CoA) and adenosine 5-triphosphate disodiumsalt (ATP). Obtained acyl-CoA is oxidized and yields 2,3-trans-Enoyl-CoAand hydrogen peroxide by the action of acyl-CoA oxidase (ACOD). In thepresence of peroxidase (POD), the hydrogen peroxide formed yields a bluepurple pigment after quantitative oxidation condensation with3-methyl-N-ethyl-N-(β-hydroxyethyl)-aniline (MEHA) and 4-aminoantipyrine(4-AA). The concentration of non-esterified fatty acids (NEFA)concentration is obtained by measuring absorbance of the blue purplecolor. NEFA standard consists of a solution of oleic acid, which is usedto produce a calibration line to calculate the amount of free fattyacids. The NEFA method is performed on the Konelab Arena 30 analyzer(Thermo Scientific, Vantaa, Finland) and the intensity of the color ismeasured at 540 nm. The reaction sample absorbance is corrected bysubtracting the absorbance of an appropriate reaction sample blank asdescribed below.

Enzyme samples were diluted to a range between 0.05-1.5 U/mL in 0.2 MTris-HCl pH 8.0. For each reaction sample and reaction sample blankglass tubes containing 50 microliter of a 0.1 M calciumchloridesolution, 500 microliter substrate solution (1% (w/v)L-alpha-phosphatidylcholine (P3556, Sigma Aldrich) in 2% Triton X-100),and 250 microliter 0.2 M Tris-HCl pH 8.0 were prepared. These werepreheated for 10 minutes in a water bath at 37° C. The reaction wasstarted by adding 100 microliter of enzyme sample to the glass tube. 10minutes after enzyme sample addition, 100 microliter of 2.0M HCl wasadded, the tube was immediately removed from the water bath and mixedwell by vortexing to terminate the reaction. For each reaction sample, acorresponding reaction sample blank was prepared by incubation ofbuffer, substrate, and calciumchloride at 37° C. After 10 minutes, 100microliter of 2.0 M HCl was added, the tube was immediately removed fromthe water bath and mixed well by vortexing. Then 100 microliter ofenzyme sample was added to the tube, and mixed well by vortexing.

Released free fatty acids were determined according to the HR SeriesNEFA-HR (2) kit instructions, which were made suitable for analyzerapplication. 150 microliter of reagent NEFA-R1 was preheated for 300seconds. Then 10 microliter of reaction sample was added, and incubationcontinued for 180 seconds. Subsequently 75 microliter of reagent NEFA-R2was added and incubation continued for 270 seconds. At this momentabsorbance was measured at 540 nm. The absorbance of each reactionsample was corrected by subtracting the absorbance of the correspondingreaction sample blank. The amount of non-esterified fatty acids wasdetermined relative to an oleic acid calibration line.

Assay 2A Galactolipase Activity at pH 5.5

As under assay 1A, except that the substrate consisted of 1% (w/v)Digalactosyldiglyceride (DGDG, plant based, from Lipid Products), in 2%Triton X-100.

Assay 2B Galactolipase Activity at pH 8.0

As under assay 1B, except that the substrate consisted of 1% (w/v)Digalactosyldiglyceride (DGDG, plant based, from Lipid Products), in 2%Triton X-100.

Assay 3A TAG-Lipase Activity at pH 5.5

As under assay 1A, except that the substrate consisted of an aqueoussolution with 4.5 mM Triolein, 1.0 M NaCl, 13% (w/v) Triton X-100(62314, Sigma Aldrich).

Assay 3B TAG-Lipase Activity at pH 8.0

As under assay 1B, except that the substrate consisted of an aqueoussolution with 4.5 mM Triolein, 1.0 M NaCl, 13% (w/v) Triton X-100(62314, Sigma Aldrich).

Assay 4A Lipolytic Activity on pNP-Linoleate at pH 5.5

Enzymatic activity of the lipolytic enzyme variant and of the parentpolypeptide (also referred as reference polypeptide) may be expressed inpNP (4-nitrophenol) Units. One Unit (U) is defined as the amount ofenzyme that liberates one micromole of 4-nitrophenol per minute underthe conditions of the test.

The principle of the assay is as follows: For the reaction sample a mixof enzyme and substrate solution is incubated at 25° C. for 30 minutes.During the incubation time, absorbance at 348_(nm) (Abs 348_(nm)) ismeasured. The initial slope (Delta Abs 348_(nm)) of the linear part ofthe absorbance measurement of the reaction sample is corrected with theinitial slope (Delta Abs 348_(nm)) of an appropriate reaction sampleblank as described below. A calibration line is measured as follows amixture of buffer, and substrate solution is incubated at 25° C. for 30minutes. During the incubation time, absorbance at 348_(nm) (Abs348_(nm)) is measured. For the calibration line the same volume ofbuffer and substrate are used as for the reaction sample but instead ofenzyme 4-nitrophenol is used). For the calibration line a range of 0-10mM 4-nitrophenol is used. The absorbance of the calibration samples isplotted against their concentration and the initial slope (Delta Abs348_(nm)) of the linear part is calculated.

The activity of the enzyme is obtained by dividing the initial slope ofthe reaction sample by the initial slope of the calibration line. Thisgives the activity of the enzyme in mM/min.

Substrate solution was prepared as follows: An 8.0 mM solution of thechromogenic substrate in 2-propanol was made. Subsequently, 3.5 mL ofthis solution was added to 46.5 mL 100 millimol/L sodium acetate bufferpH 5.5 containing 1% Triton X-100, under vigorous stirring. Substratewas pNP-linoleate (>95% pure, from Syncom, The Netherlands).

Enzyme was diluted in 100 millimol/L sodium acetate buffer pH 5.5containing 1% Triton X-100, such that the absorbance increase after 30minutes is less than 1.0. Reaction was started by mixing 10 microliterof diluted enzyme sample with 240 microliter of substrate solution in amicrotiter plate. 200 microliter of this mixture was added to a newmicrotiter plate, placed in a TECAN Infinite M1000 micro titer platereader, temperature is kept at 25° C., and the change in absorbance ofthe mixture was measured for 30 minutes at 348 nm (isosbestic point of4-nitrophenol). The reaction sample blank was prepared by adding 10microliter of sodium acetate buffer pH 5.5 instead of enzyme sample tothe substrate solution and then following the steps identical asdescribed above for the enzyme reaction. A calibration line was producedfrom 4-nitrophenol dissolved in 100 millimol/L sodium acetate buffer pH5.5 containing 1% Triton X-100.

The absorbance of the reaction samples was plotted against the time. Theslope was determined over the initial linear part of the absorbancemeasurement. The initial slope of the reaction samples was corrected bysubtracting the slope of the reaction sample blank. Subsequentlyactivity was calculated relative to the slope of the calibration line.

Assay 4B Lipolytic Activity on pNP-Linoleate at pH 7.0

As under assay 4A, except that the buffer consisted of 100 millimol/LMOPS pH 7.0 containing 1% Triton X-100.

Assay 5A Lipolytic Activity on pNP-Oleate at pH 5.5

As under assay 4A, except that the substrate consisted of pNP-oleate(>95% pure, from Syncom, The Netherlands).

Assay 5B Lipolytic Activity on pNP-Oleate at pH 7.0

As under assay 4B, except that the substrate consisted of pNP-oleate(>95% pure, from Syncom, The Netherlands).

Assay 6A Lipolytic Activity on pNP-Stearate at pH 5.5

As under assay 4A, except that the substrate consisted of pNP-stearate(N3627, Sigma Aldrich).

Assay 6B Lipolytic Activity on pNP-Stearate at pH 7.0

As under assay 4B, except that the substrate consisted of pNP-stearate(N3627, Sigma Aldrich).

Assay 7A Lipolytic Activity on pNP-Butyrate at pH 5.5

As under assay 4A, except that the substrate consisted of pNP-butyrate(N9876, Sigma Aldrich).

Assay 7B Lipolytic Activity on pNP-Butyrate at pH 7.0

As under assay 4B, except that the substrate consisted of pNP-butyrate(N9876, Sigma Aldrich).

Assay 8A Lipolytic Activity on pNP-Palmitate at pH 8.5

As under assay 4A, except that the buffer consisted of 100 millimol/LTris pH 8.5 containing 1% Triton X-100, and the substrate consisted ofpNP-palmitate (N2752, Sigma Aldrich).

Assay 9 Lipolytic Activity pH Profile on pNP-Linoleate

As under assay 4A, except that the buffer consisted of either 100millimol/L sodium acetate buffer pH 4.0 containing 1% Triton X-100, 100millimol/L sodium acetate buffer pH 4.5 containing 1% Triton X-100, 100millimol/L sodium acetate buffer pH 5.0 containing 1% Triton X-100, 100millimol/L sodium acetate buffer pH 5.5 containing 1% Triton X-100millimol/L, 100 millimol/L MES buffer pH 6.0 containing 1% Triton X-100,100 millimol/L MOPS buffer pH 6.5 containing 1% Triton X-100 millimol/L,100 millimol/L MOPS buffer pH 7.0 containing 1% Triton X-100 millimol/L,100 millimol/L MOPS buffer pH 7.5 containing 1% Triton X-100 millimol/L,or 100 millimol/L TRIS buffer pH 8.0 containing 1% Triton X-100millimol/L.

Determination of Altered Properties

Altered properties of lipolytic enzyme variants according to thedisclosure as compared with a reference polypeptide having an amino acidsequence as set out in amino acids 34 to 304 of SEQ ID NO: 2 wereobtained as follows.

Firstly, the properties of the variants and the reference polypeptidewere measured as described under Assay 1A to Assay 9 above.

Secondly, from these measurements the percentages (%) listed in Tables 2to 17 below were obtained. The way to obtain the percentages listed inthe tables below is explained via an exemplary determination andcalculation of galactolipase to TAG-lipase activity ratio (Table 2). The% in Tables 3 to 17 were obtained analogously.

Exemplary Determination and Calculation of Galactolipase to TAG-LipaseActivity Ratio of Variant #

The activity of variant # on Digalactosyldiglyceride (DGDG) (measured asdescribed under Assay 2A above) was expressed as a ratio to the activitymeasured for the same variant on triolein (measured as described underAssay 3A above).

The reference polypeptide (having an amino acid sequence as set out inamino acids 34 to 304 of SEQ ID NO: 2) was subjected to the sameexperimental conditions.

If the activity of variant # would be 850 units/mL on DGDG (Assay 2A),and 1000 units/ml on triolein (Assay 3A), the ratio of activity ofgalactolipase to TAG-lipase for variant # would be 0.85.

If the activity of the reference polypeptide would be 600 units/mL onDGDG (Assay 2A), and 1000 units/mL on triolein (Assay 3A), then theratio of the activity of galactolipase to TAG-lipase for the referencepolypeptide would be 0.6.

This value of the reference polypeptide is then normalized to 100%.

In this exemplary calculation the galactolipase to TAG-lipase activityratio of variant # compared with the reference polypeptide would then be(0.85/0.60)×100%=142%. 142% (for variant #) is an increase compared with100% (for the reference polypeptide), as a result variant # is said tohave an increased galactolipase to TAG-lipase activity ratio comparedwith the reference polypeptide. This value of 142% is thus a normalised%. In the tables in the examples normalised % are listed.

In short table 2 lists: the ratio of

[Activity of variant # in Assay 2A]: [Activity of variant # in assay3A],

expressed as a percentage of the ratio of

[Activity of the reference polypeptide in Assay 2A]: [Activity of thereference polypeptide in Assay 3A].

The percentage thus obtained is the galactolipase to TAG-lipase activityratio as listed in table 2.

Example 1. Design and Cloning of the Lipolytic Enzyme Variant of theDisclosure

The protein sequence (amino acid sequence) of the reference polypeptide(also referred to as parent polypeptide) having lipolytic activity isshown in amino acids 34 to 304 SEQ ID NO: 2. A codon-adapted DNAsequence for expression of the lipolytic enzyme proteins (lipolyticenzyme variants and reference polypeptide) in Aspergillus niger wasdesigned containing additional BsaI type II restriction enzyme sites toenable subcloning in the Aspergillus expression vector pGBFIN-50 (seealso FIG. 1). Codon adaptation was performed as described inWO2008/000632. The codon optimized DNA sequence for expression of thegene encoding the reference polypeptide in A. niger is shown in SEQ IDNO: 1.

The translational initiation sequence of the glucoamylase glaA promoterwas modified into 5′-CACCGTCAAA ATG-3′ (SEQ ID NO: 3) (already presentin the Aspergillus expression vector pGBFIN-50) and an optimaltranslational termination sequence 5′-TAAA-3′ was used in the generationof the lipolytic enzyme expression constructs (as also detailed inWO2006/077258 and WO2011/009700). The DNA sequences coding for thelipolytic enzyme variants and for the reference polypeptide of theinvention were synthesized completely (DNA2.0, Menlo Park, USA) andcloned into Aspergillus niger expression vector pGBFIN-50 thrurepetitive steps of BsaI digestion and ligation (GoldenGate cloningmethod (New England Biolabs), according standard procedure. Theresulting vectors containing the lipolytic enzyme expression cassette(including the expression cassette for the lipolytic enzyme variants andthe reference polypeptide see Table 1 below for details) under controlof the glucoamylase promoter and were named pGBFINPL-00 up topGBFINPL-51.

Subsequently, A. niger GBA 306 was transformed with a PCR-amplifiedPgla-3′gla fragment generated using the pGBFINPL-00 up to pGBFINPL-51vectors as template. The PCR fragment is comprising the lipolytic enzymeexpression cassette under control of the glucoamylase promoter andterminator as well as the hygromycin selection marker. Alternatively, aNotI-digested and purified fragment of the pGBFINPL-00 up to pGBFINPL-51vectors constructed, containing the lipolytic enzyme expression cassetteand the hygromycin selection marker could have been used. Transformationexperiments were performed with strain and methods as described inWO199846772, WO199932617, WO2011009700, WO2012001169, WO2013135729,WO2014013073 and WO2014013074 and references therein. Aftertransformation, the protoplasts were plated onto selective regenerationmedium consisting of Aspergillus minimal medium supplemented with 60μg/mL Hygromycin B. After incubation for 5-10 days at 30° C., singletransformants were restreaked to single colonies on PDA (Potato DextroseAgar) supplemented with 60 μg/mL Hygromycin B. After 5-7 days growth andsporulation at 30° C., single colonies were transferred to PDA plates.Following growth for 5-7 days at 30° C. spores were isolated and used asinoculation material for shake flask fermentations.

Transformants of lipolytic enzymes named PL-00, PL-01 up to andincluding PL-51, were used for fermentations (see Table 1 below fordetails)

The amino acid changes that were introduced in the 51 lipolytic enzymevariants are listed in Table 1.

TABLE 1 Amino acid changes introduced in the parent polypeptide, whereinthe parent polypeptide has an amino acid sequence as set out in aminoacids 34 to 304 of SEQ ID NO: 2. Amino acids are depicted according tothe single letter annotation. # Amino acid change* 00 (parentpolypeptide also referred None to as reference polypeptide) 01 Y53F 02Y53S 03 S112T 04 I113H 05 I113L 06 I113N 07 I113R 08 I113T 09 N117D 10N121D 11 L122A 12 L122M 13 F124L 14 H138E 15 H138S 16 F141M 17 F141Y 18A178G 19 V179L 20 V179M 21 V179S 22 L182F 23 G200A 24 P202A 25 R203M 26P229Q 27 P235L 28 F238L 29 L282E 30 L282F 31 L282K 32 L282M 33 L282N 34L282R 35 L282S 36 L282T 37 I284A 38 I284D 39 I284E 40 I284F 41 I284M 42I284N 43 I284P 44 I284Q 45 I284S 46 I284T 47 A286L 48 D295E 49 D295G 50D295N 51 D295S *positions being defined with reference to SEQ ID NO: 2

Example 2. Expression of the Lipolytic Enzyme Variants and the ReferencePolypeptide Using the PL-00 to PL-51 Transformants from Example 1

Fresh A. niger PL-00 up to PL-51 spores were prepared and used forgenerating lipolytic enzyme sample material by cultivation of thestrains in shake flask. A. niger strains were precultured in 20 mLpreculture medium in a 100 mL shake flask with baffle containing perliter: 100 g Corn Steep Solids (Roquette), 1 g NaH2PO4.H2O, 0.5 gMgSO4.7H2O, 10 g Glucose.H2O, 0.25 g Basildon pH5.8. After overnightgrowth at 34° C. and 170 rpm 10 mL of this culture was transferred to100 mL fermentation medium in 500 mL shake flasks with baffle.Fermentation medium contains per liter: (150 g maltose, 60 gbacto-soytone, 15 g (NH₄)₂SO₄, 1 g NaH₂PO₄.H₂O, 1 g MgSO₄.7H₂O, 1 gL-arginine, 0.08 g Tween-80, 0.02 g Basildon, 20 g MES, pH 6.2 Cultureswere grown for 2-7 days at 34° C., 170 rpm. Culture supernatants wererecovered by centrifugation for 10 min at 5000×g.

Measurement of Lipolytic Activity

Enzymatic activity of the lipolytic enzyme variants and of the parentpolypeptide (also referred as reference polypeptide) may be expressed inNEFA Units. One Unit (U) is defined as the amount of enzyme thatliberates one micromole of free fatty acid per minute under the definedassay conditions.

The lipolytic activity of the lipolytic enzyme variants and of theparent polypeptide having lipolytic activity was demonstrated using theAssays described herein (see examples below). All lipolytic enzymevariants listed in Table 1 and the parent polypeptide showed lipolyticactivity.

Example 3: Activity Ratio of Activity on pNP-Stearate to Activity onpNP-Butyrate at pH 5.5 of Variants According to the Disclosure

Activity ratio of activity on pNP-stearate to activity on pNP-butyrateat pH 5.5 of variants as compared to the reference polypeptide wasdetermined using the enzymes obtained in Example 2. This ratio wasdetermined as described herein under Materials and Methods above(applying Assay 6A, Assay 7A, and calculating analogous to “Exemplarydetermination and calculation of galactolipase to TAG-lipase activityratio of variant #”). The results are listed in Table 2.

TABLE 2 Activity ratio of activity on pNP-stearate to activity onpNP-butyrate at pH 5.5 of variants according to the disclosure comparedto the reference polypeptide. The activity on pNP-stearate to activityon pNP-butyrate of the reference polypeptide (polypeptide having anamino acid sequence as set out in amino acids 34 to 304 of SEQ ID NO:2), was set at 100%. In the table normalised % are listed. An activityratio of more than 100% shows that the variant has an increased activityon pNP-stearate compared to the reference polypeptide. Amino AcidActivity ratio of activity on pNP-stearate Change* to activity onpNP-butyrate at pH 5.5 (%) Y53S >500% R203M  215% *positions beingdefined with reference to SEQ ID NO: 2

Example 4: Activity Ratio of Activity on pNP-Stearate to Activity onpNP-Butyrate at pH 7.0 of Variants According to the Disclosure

Activity ratio of activity on pNP-stearate to activity on pNP-butyrateat pH 7.0 of variants as compared to the reference polypeptide wasdetermined using the enzymes obtained in Example 2. This ratio wasdetermined as described herein under Materials and Methods above(applying Assay 6B, Assay 7B, and calculating analogous to “Exemplarydetermination and calculation of galactolipase to TAG-lipase activityratio of variant #”). The results are listed in Table 3.

TABLE 3 Activity ratio of activity on pNP-stearate to activity onpNP-butyrate at pH 7.0 of variants according to the disclosure comparedto the reference polypeptide. The activity on pNP-stearate to activityon pNP-butyrate of the reference polypeptide (polypeptide having anamino acid sequence as set out in amino acids 34 to 304 of SEQ ID NO:2), was set at 100%. In the table normalised % are listed. An activityratio of more than 100% shows that the variant has an increased activityon pNP-stearate compared to the reference polypeptide. Amino AcidActivity ratio of activity on pNP-stearate Change* to activity onpNP-butyrate at pH 7.0 (%) Y53S >500%  A178G 198% V179M 181% P202A 233%R203M >500%  L282F 309% I284Q 241% *positions being defined withreference to SEQ ID NO: 2

Example 5: Activity Ratio of Activity on pNP-Oleate to Activity onpNP-Butyrate at pH 5.5 of Variants According to the Disclosure

Activity ratio of activity on pNP-oleate to activity on pNP-butyrate atpH 5.5 of variants as compared to the reference polypeptide wasdetermined using the enzymes obtained in Example 2. This ratio wasdetermined as described herein under Materials and Methods above(applying Assay 5A, Assay 7A, and calculating analogous to “Exemplarydetermination and calculation of galactolipase to TAG-lipase activityratio of variant #”). The results are listed in Table 4.

TABLE 4 Activity ratio of activity on pNP-oleate to activity onpNP-butyrate at pH 5.5 of variants according to the disclosure comparedto the reference polypeptide. The activity on pNP-oleate to activity onpNP-butyrate of the reference polypeptide (polypeptide having an aminoacid sequence as set out in amino acids 34 to 304 of SEQ ID NO: 2), wasset at 100%. In the table normalised % are listed. An activity ratio ofmore than 100% shows that the variant has an increased activity onpNP-oleate compared to the reference polypeptide. Amino Acid Activityratio of activity on pNP-oleate Change* to activity on pNP-butyrate atpH 5.5 (%) Y53S 498% S112T 142% A178G 131% V179M 133% R203M 175%*positions being defined with reference to SEQ ID NO: 2

Example 6: Activity Ratio of Activity on pNP-Oleate to Activity onpNP-Butyrate at pH 7.0 of Variants According to the Disclosure

Activity ratio of activity on pNP-oleate to activity on pNP-butyrate atpH 7.0 of variants as compared to the reference polypeptide wasdetermined using the enzymes obtained in Example 2. This ratio wasdetermined as described herein under Materials and Methods above(applying Assay 5B, Assay 7B, and calculating analogous to “Exemplarydetermination and calculation of galactolipase to TAG-lipase activityratio of variant #”). The results are listed in Table 5.

TABLE 5 Activity ratio of activity on pNP-oleate to activity onpNP-butyrate at pH 7.0 of variants according to the disclosure comparedto the reference polypeptide. The activity on pNP-oleate to activity onpNP-butyrate of the reference polypeptide (polypeptide having an aminoacid sequence as set out in amino acids 34 to 304 of SEQ ID NO: 2), wasset at 100%. In the table normalised % are listed. An activity ratio ofmore than 100% shows that the variant has an increased activity onpNP-oleate compared to the reference polypeptide. Amino Acid Activityratio of activity on pNP-oleate Change* to activity on pNP-butyrate atpH 7.0 (%) Y53S >500%  S112T 139% F141M 164% A178G 165% V179L 153% V179M165% R203M >500%  P235L 167% L282F 319% *positions being defined withreference to SEQ ID NO: 2

Example 7: Activity Ratio of Activity on pNP-Linoleate to Activity onpNP-Butyrate at pH 5.5 of Variants According to the Disclosure

Activity ratio of activity on pNP-linoleate to activity on pNP-butyrateat pH 5.5 of variants as compared to the reference polypeptide wasdetermined using the enzymes obtained in Example 2. This ratio wasdetermined as described herein under Materials and Methods above(applying Assay 4A, Assay 7A, and calculating analogous to “Exemplarydetermination and calculation of galactolipase to TAG-lipase activityratio of variant #”). The results are listed in Table 6.

TABLE 6 Activity ratio of activity on pNP-linoleate to activity on pNP-butyrate at pH 5.5 of variants according to the disclosure compared tothe reference polypeptide. The activity on pNP-linoleate to activity onpNP-butyrate of the reference polypeptide (polypeptide having an aminoacid sequence as set out in amino acids 34 to 304 of SEQ ID NO: 2), wasset at 100%. In the table normalised % are listed. An activity ratio ofmore than 100% shows that the variant has an increased activity onpNP-linoleate compared to the reference polypeptide. Amino Acid Activityratio of activity on pNP-linoleate Change* to activity on pNP-butyrateat pH 5.5 (%) Y53S >500%  S112T 172% F141M 157% A178G 168% V179L 142%V179M 167% L182F 152% R203M 185% P235L 165% *positions being definedwith reference to SEQ ID NO: 2

Example 8: Activity Ratio of Activity on pNP-Linoleate to Activity onpNP-Butyrate at pH 7.0 of Variants According to the Disclosure

Activity ratio of activity on pNP-linoleate to activity on pNP-butyrateat pH 7.0 of variants as compared to the reference polypeptide wasdetermined using the enzymes obtained in Example 2. This ratio wasdetermined as described herein under Materials and Methods above(applying Assay 4B, Assay 7B, and calculating analogous to “Exemplarydetermination and calculation of galactolipase to TAG-lipase activityratio of variant #”). The results are listed in Table 7.

TABLE 7 Activity ratio of activity on pNP-linoleate to activity on pNP-butyrate at pH 7.0 of variants according to the disclosure compared tothe reference polypeptide. The activity on pNP-linoleate to activity onpNP-butyrate of the reference polypeptide (polypeptide having an aminoacid sequence as set out in amino acids 34 to 304 of SEQ ID NO: 2), wasset at 100%. In the table normalised % are listed. An activity ratio ofmore than 100% shows that the variant has an increased activity onpNP-linoleate compared to the reference polypeptide. Amino Acid Activityratio of activity on pNP-linoleate Change* to activity on pNP-butyrateat pH 7.0 (%) Y53S >500%  A178G 154% V179M 490% R203M >500%  L282F 341%*positions being defined with reference to SEQ ID NO: 2

The invention claimed is:
 1. A variant polypeptide having lipolyticactivity, wherein the variant has an amino acid sequence which, whenaligned with the amino acid sequence as set out in SEQ ID NO: 2,comprises at least one substitution of an amino acid residue at aposition corresponding to any of the positions 53, 112, 141, 178, 179,182, 202, 203, 235, 282, 284, said positions being defined withreference to SEQ ID NO: 2, and wherein said variant has at least 75%identity with the mature polypeptide of SEQ ID NO:
 2. 2. The variantpolypeptide according to claim 1, wherein the one or more of the aminoacid substitutions is selected from the group consisting of Y53S S112TF141M A178G V179L V179M L182F P202A R203M P235L L282F and I284Q.
 3. Thevariant according to claim 1, wherein the variant includes an increasedratio of lipolytic activity on esters of long chain fatty acids:lipolytic activity on esters of short chain fatty acids as compared tothe lipolytic activity of a reference polypeptide measured under thesame conditions.
 4. A nucleic acid sequence encoding the variantpolypeptide according to claim
 1. 5. A nucleic acid construct comprisingthe nucleic acid sequence of claim 4 operably linked to one or morecontrol sequences capable of directing the expression of a lipolyticenzyme in a suitable expression host.
 6. A recombinant host cellcomprising a recombinant expression vector comprising the nucleic acidconstruct of claim
 5. 7. A method for producing a lipolytic polypeptidevariant of claim 1 comprising cultivating a recombinant host cellcomprising a nucleic acid encoding the variant polypeptide underconditions conducive to production of the lipolytic enzyme variant andrecovering the lipolytic enzyme variant.
 8. A method of producing alipolytic polypeptide variant having an increased ratio of lipolyticactivity on esters of long chain fatty acids: lipolytic activity onesters of short chain fatty acids as compared to the lipolytic activityof reference polypeptide measured under the same conditions, whichmethod comprises: a) selecting a polypeptide having lipolytic activity;b) substituting at least one amino acid residue at a positioncorresponding to any of the positions 53, 112, 141, 178, 179, 182, 202,203, 235, 282, 284, said positions being defined with reference to SEQID NO: 2; c) optionally substituting one or more further amino acids asdefined in b), wherein the resulting polypeptide has at least 75%identity with the mature polypeptide of SEQ ID NO: 2; d) preparing thevariant resulting from a)-c); e) determining the lipolytic activity onesters of long chain fatty acids and the lipolytic activity on esters ofshort chain fatty acids of the variant; and f) selecting a varianthaving an increased ratio of lipolytic activity on esters of long chainfatty acids: lipolytic activity on esters of short chain fatty acids ascompared to the lipolytic activity of the reference polypeptide measuredunder the same conditions.
 9. A composition comprising the variantpolypeptide according to claim 1 and one or more components selectedfrom the group consisting of milk powder, gluten, granulated fat, anadditional enzyme, an amino acid, a salt, an oxidant, a reducing agent,an emulsifier, sodium stearoyl lactylate, calcium stearoyl lactylate,polyglycerol esters of fatty acids and diacetyl tartaric acid esters ofmono- and diglycerides, a gum, a flavour, an acid, a starch, a modifiedstarch, a humectant and a preservative.
 10. A dough comprising thevariant polypeptide according to claim
 1. 11. A process for productionof a dough comprising combining an effective amount of the variantpolypeptide according to claim 1 with at least one dough ingredient. 12.A process for production of a baked product, which method comprisesbaking the dough according to claim
 10. 13. The variant according toclaim 2, wherein the variant includes an increased ratio of lipolyticactivity on esters of long chain fatty acids: the lipolytic activity onesters of short chain fatty acids as compared to the lipolytic activityof the reference polypeptide of SEQ ID NO: 2 having lipolytic activitymeasured under the same conditions.
 14. The variant according to claim13, wherein the lipolytic activity on long chain fatty acids is measuredas lipolytic activity on pNP-linoleate at pH 5.5 according to assay 4A,which method comprises: a) diluting the reference or variant polypeptidein 100 millimol/L sodium acetate buffer pH 5.5 containing 1% TritonX-100 to a concentration where the absorbance at 348 nm is less than 1.0after 30 minutes; b) preparing a substrate solution comprising 3.5 mL ofan 8.0 M solution of chromogenic pNP-lineolate in 2-proponol and 46.5 mlof 100 mmol/L sodium acetate buffer pH 5.5, 1% Triton X-100; c) adding10 μl of diluted enzyme from a) to 240 μl substrate solution of b) andincubating at 25° C.; d) measuring the absorbance over 30 minutes at 358nm and determining the slope; and wherein the activity on short chainfatty acids is measured as lipolytic activity on pNP-butyrate at pH 5.5according to assay 7A which method comprises a)-d), except that thesubstrate in b) is pNP-butyrate at pH 5.5.
 15. The method according toclaim 8, wherein the lipolytic activity on long chain fatty acids ismeasured as lipolytic activity on pNP-linoleate at pH 5.5. according toassay 4A, which method comprises: a) diluting the reference or variantpolypeptide in 100 millimol/L sodium acetate buffer pH 5.5 containing 1%Triton X-100 to a concentration where the absorbance at 348 nm is lessthan 1.0 after 30 minutes; b) preparing a substrate solution comprising3.5 mL of an 8.0 M solution of chromogenic pNP-lineolate in 2-proponoland 46.5 ml of 100 mmol/L sodium acetate buffer pH 5.5, 1% Triton X-100;c) adding 10 μl of diluted enzyme from a) to 240 μl substrate solutionof b) and incubating at 25° C.; d) measuring the absorbance over 30minutes at 358 nm and determining the slope; and wherein the activity onshort chain fatty acids is measured as lipolytic activity onpNP-butyrate at pH 5.5 according to assay 7A which method comprisesa)-d), except that the substrate in b) is pNP-butyrate at pH 5.5.
 16. Acomposition comprising the variant polypeptide according to claim 2, andone or more components selected from the group consisting of milkpowder, gluten, granulated fat, an additional enzyme, an amino acid, asalt, an oxidant, a reducing agent, an emulsifier, sodium stearoyllactylate, calcium stearoyl lactylate, polyglycerol esters of fattyacids and diacetyl tartaric acid esters of mono- and diglycerides, agum, a flavour, an acid, a starch, a modified starch, a humectant and apreservative.
 17. The variant according to claim 1, wherein thelipolytic activity on long chain fatty acids is measured as lipolyticactivity on pNP-linoleate at pH 5.5. according to assay 4A, which methodcomprises: a) diluting the reference or variant polypeptide in 100millimol/L sodium acetate buffer pH 5.5 containing 1% Triton X-100 to aconcentration where the absorbance at 348 nm is less than 1.0 after 30minutes; b) preparing a substrate solution comprising 3.5 mL of an 8.0 Msolution of chromogenic pNP-lineolate in 2-proponol and 46.5 ml of 100mmol/L sodium acetate buffer pH 5.5, 1% Triton X-100; c) adding 10 μl ofdiluted enzyme from a) to 240 μl substrate solution of b) and incubatingat 25° C.; d) measuring the absorbance over 30 minutes at 358 nm anddetermining the slope; and wherein the activity on short chain fattyacids is measured as lipolytic activity on pNP-butyrate at pH 5.5according to assay 7A which method comprises a)-d), except that thesubstrate in b) is pNP-butyrate at pH 5.5.
 18. A dough comprising thevariant polypeptide according to claim
 2. 19. A process for productionof a dough, comprising combining an effective amount of the variantpolypeptide according to claim 2 with at least one dough ingredient.